(752c) Butanol Production by Clostridium acetobutylicum in a Series of Packed Bed Biofilm Reactors

Raganati, F., Università degli Studi di Napoli Federico II
Olivieri, G., Università degi Studi di Napoli Federico II
Salatino, P., Università degli Studi di Napoli Federico II
Marzocchella, A., Università degi Studi di Napoli Federico II

The development of biotechnological processes to produce butanol
from renewable resources as eco-sustainable alternatives to the petrochemical production
routes (Kumar et al., 2012) is still an open challenge. Acetone-Butanol-Ethanol
(ABE) fermentation by clostridia is drawing new interest as a way to turn
renewable resources into valuable base chemicals and liquid fuels. Nevertheless
the high industrial potential interest for the butanol production by the
biotechnological route, some features of the ABE fermentation process hinder
its success on an industrial scale. Indeed, the ABE fermentation is
characterized by low yield, the acid-solvent two phase feature, and low
concentration of butanol due to its inhibiting effect on fermentation. Moreover,
the cost of traditional feedstocks - starch and molasses – is a larger fraction
of the butanol sale price. Therefore, the use of a low-cost renewable resource
is a pre-requisite for the industrial success of biotechnological process to
produce butanol. Key issues for the success of the ABE fermentation process
include the detailed metabolic analysis of the clostridia coupled with the
development of improved engineered strains too (Papoutsakis 2008). 

Reactor design and operating conditions play a key role in
fermentative productions. The main factors that hinder the commercial development
of the traditional ABE batch fermentation processes include low cell density,
cell – and relative feedstock - lost at the end of the fermentation, low
reactor productivity, high down-times, nutritional limitations and severe
product inhibition (Chen and Blaschek, 1999). Process intensification may be
obtained by increasing cell concentration in the reactor: cell immobilized
reactors and retention membrane reactors are two potential solutions (Qureshi
et al., 2005). These reactors configuration takes also the advantage to be
operated continuously (Qureshi et al., 2000; Procentese et al., 2015).
Continuous bioconversions are characterized by several advantages with respect
to batch cultures in biofilm reactors (Qureshi et al., 2000). The main advantages
are: the high cell concentration, the reactor operation at high dilution rates –
high throughout - without cell washout, high yield, high butanol concentration
that enhance butanol recovery performances. Moreover, the biofilm support can
be reused.

The reactor design and its optimization for ABE fermentation may
take advantages form reactor modelling The performance of ABE fermentation
process, in terms of selectivity and yield of solvents, depends on several physical
and design parameters, on the Clostridium strain, and on the mode of
fermentation operation. Simulation of the fermentation process with
mathematical models gives an insight into the characteristics of the process,
and also helps to identify the influence of each variable on the overall process.

This contribution reports recent advance on ABE fermentation at
Napoli. The study regarded an innovative immobilized cell reactor system: an
experimental campaign and the development of a model.

The anaerobic solventogenic commercial Clostridium acetobutylicum
DSM 792 was used for the fermentation process. The conversion was carried out
in 4 packed bed biofilm reactors (PBBRs) connected in series: the first reactor
(fed with stream bearing the the carbon source) was operated under acidogenesis
conditions, and the three successive reactors were operated under
solventogenesis conditions. The two phases of the ABE fermentation were
operated in separate vessels: acids were produced in first section of the
system, the produced acids and the residual sugar were converted in solvents in
the second section. The PBBR system performance was characterized in terms of
final butanol concentration and productivity as a function of the dilution

A mathematical model of the PBBRs system was formulated using glucose
kinetic data assessed experimentally (Procentese et al. 2015a and 2015b). The
proposed model was an unstructured-unsegregated model and summarizes
biochemical as well as physiological issues of growth and metabolite synthesis
by the production strain. The key fermentation rates were expressed and
evaluated with regard to substrate consumption and butanol end-product
inhibitory effects.





The apparatus was made of reactor system, liquid pumps, heating
apparatus, device for pH control, and on-line diagnostics (sketch in Fig. 1).
The reactor system was made of four fixed beds. Each bed was at the bottom of a
100 mL glass lined pipe (4 cm ID, 8 cm high) jacketed for the heat exchange.
Water from an external circulating water bath (Julabo heating circulator MA4)
was fed into the jacket of each reactor to keep the operating temperature at 37
°C. The liquid head was controlled by the overflow duct in each reactor: the
working volume of each reactor was set at 40 mL. Nitrogen was sparged at the
bottom of each reactor to ensure anaerobic conditions. The pH control device –
one for each reactor - included a pH-meter, a peristaltic pump, a vessel with NaOH
0.3 M solution and a controller.

Tygon rings (3/1 mm OD/ID) were chosen as biofilm carriers.

Clostridium acetobutylicum DSM 792 was
used. Details regarding the reactivation and pre-inoculum procedures are
reported in Raganati et al. 2013.

The composition of the medium fed to the PBBR system is reported in (Procentese
et al. 2015a). Glucose at 100. g/L was used as carbon source.



Fig.1: Outline of the
apparatus used for the continuous process: A) PBBRs connected in parallel
during the start-up phase; B) PBBRs connected in series during the butanol
production phase. b: pH measure/control device.


Two PBBR configurations were used.

Parallel configuration) The four fixed
beds were operated in parallel (Fig. 1A). This configuration was used during
start-up to promote biofilm formation.

Series configuration) The four fixed
beds were connected in series (Fig. 1B). This configuration was used during
butanol production after a biofilm layer had formed in each unit in parallel

Butanol production tests were carried out with the 4 PBBRs connected
in series (Fig.1B) and operated at pre-set conditions. The pH of the reactor 1 (Fig.1B)
was set at 5.5 to promote acidogenesis conditions. The pH of the reactors 2, 3,
and 4 (Fig.1B) was set at 4.7 to promote solventogenesis conditions.

The overall dilution rate (D) – ratio between the feeding flow rate
and the total volume of the 4 fixed beds – ranged between 0.05 and 1.4 h-1.
After setting the dilution rate, the reactor system was operated until steady
state conditions were reached: metabolite and glucose concentration in each
reactor staying constant for at least 5 times the space-time of the reactor.

The PBBRs system was successfully operated for more than three
months to produce butanol. The reactor performances – butanol productivity and
butanol concentration – were assessed as a function of the reactor system
dilution rate. By tuning the D it was possible: i) to totally convert the
carbon source (D <0.15 h-1); ii) to maximize the concentration of
butanol in the produced stream (about 15 g/L at D = 0.65 h-1); iii)
to maximize the butanol productivity (about 12.6 g/Lh at D = 0.9 h-1).


The PBBRs system was simulated by an unstructured-unsegregated model
that summarizes biochemical as well as physiological aspects of growth and
metabolite synthesis.

Kinetic data regarding the growth rate (acidogenesis) and the
butanol production rate (solventogenesis) were from specific experimental
campaigns (details in Procentese et al. 2015a and 2015b). The main assumptions of
the proposed model are reported hereinafter.

the PBBR system was assumed as a series of CSTR;

the biomass present in each PBBR as free cells
and immobilized cells was a heterogeneous cell population consisting of:
acidogenic cells, solventogenic cells and spores;

the kinetics of cell growth and butanol
production of biofilm-cells were assumed equal to those of the free cells;

cells attachment and detachment processes were

The metabolites/sugar profiles and cell distribution within the
biofilm layer in the PBBR series were assessed as a function of the dilution
rate and of the sugar composition in the recator system feeding.

The comparison of the theoretical results and of the continuous
fermentation tests was promising.


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