Butanol Production by Clostridium acetobutylicum in a Series of Packed Bed Biofilm Reactors

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 rate.

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.

 

 

 

PBBR SYSTEM

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 mode.

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).

MODEL

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 considered.

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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