Zeolite as a carrier for lactic acid bacteria in biorefinery processes

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1 Zeolite as a carrier for lactic acid bacteria in biorefinery processes Aleksandra Djukić-Vuković 1 *, Ljiljana Mojović 1, Jelena Pejin 2, Sunčica Kocić-Tanackov 2 *adjukic@tmf.bg.ac.rs 1 Faculty of Technology and Metallurgy, University of Belgrade 2 Faculty of Technology, University of Novi Sad

2 Zeolite as a carrier? Natural zeolite is mechanically stable, inert aluminosilicate with high capacity for adsorption and exchange of cations. Zeolites could be designed for specific purposes. Zeolites are often used as an additive in detergents and molecular sieves are implemented in petrol industry for removal of water during the oil processing (hydrocracking). Type X has a basic structure with exchangeable sodium ion.

3 Lactic acid production on stillage? Lactic acid is widely used chemical in food, pharmaceutical and chemical industry 7% annual growth rate. High demand for lactic acid is driven by increased production of polidilactides - biocompatible polymers used for medical, pharmaceutical and food industry purposes. Industrially, lactic acid fermentations were mostly performed in batch processes on synthetic substrates often with nitrogen source supplementation. Fermentation enables selective production of L or D isomer.

4 Lactic acid production on stillage? Biorefinery concept enables utilization of cheap and abundant substrates like stillage and if high productivity of lactic acid could be achieved - low price of lactic acid with reduction of wastewater treatment costs. Distillery stillage is a by-product of bioethanol production on different starch feedstocks. In this study, liquid fraction of stillage from bioethanol production on wasted bread was utilized. Stillage is nitrogen opulent substrate and it was previously shown that stillage is suitable substrate for growth of lactic acid bacteria (Lactobacillus rhamnosus ATCC 7469).

5 Material and methods Inoculum- Lactobacillus rhamnosus ATCC 7469 immobilized onto powdered 13X Na-zeolite molecular sieves (Sigma-Aldrich ) by adsorption Inoculum preparation The 10% MRS overnight culture of L. rhamnosus ATCC 7469 was centrifuged (10,000 rpm, 5 min), twice washed with sterile 0.8% (w/v) NaCl solution and the biomass was suspended in 200 ml of fresh MRS broth with addition of 2% (w/v) powdered Na-zeolite. After 12 h incubation at 41 C, the culture was centrifuged (1000 rpm, 5 min), supernatant with free cells was thrown and the sediment of L. rhamnosus ATCC 7469 cells adsorbed onto zeolite was washed with sterile 0.8% (w/v) NaCl solution and used as an inoculum for fermentation. The morphology of the zeolite with immobilized L. rhamnosus ATCC 7469 was studied by field emission scanning electron microscopy (FESEM) TESCAN Mira3 XMU at 20 kv.

6 Material and methods Fermentation media - sterilized liquid wasted bread stillage from bioethanol plant Reahem, Serbia, initial sugar concentration was set at approximately 50 g /L, repeated batch strategy was performed. Fermentation 41 C, gas-pack system with Anaerocult C bags with shaking of 90 rpmmicroaerophylic, ph control: addition of 30% NaOH solution in 4 hours intervals, recirculation after decrease in sugar concentration in media below 20 g /L. During the fermentation: number of cells (pour plate counting), sugar concentration (DNS method), lactic acid concentration (L-/D lactic acid enzyme assay Megazyme, Ireland).

7 Immobilization of L. rhamnosus ATCC 7469 onto zeolite Porous zeolite with alkaline structure - Lewis acid L. rhamnosus surface - Lewis bases Basic structure- additional ph control Adsorption capacity for biopolymers exopolysaccharides of L. rhamnosus Advantages of immobilized system Immobilization enables recirculation of cells and easier separation Commonly a part of animal diet - positive effect on animal health - adsorption capacity

8 Sugar concentration (g L -1 ) Lactic acid concentration (g L -1 ) Sugar concentration (g L -1 ) Lactic acid concetration (g L -1 ) Zeolite as a carrier for lactic acid bacteria in biorefinery processes Repeated batch fermentation with L. rhamnosus immobilized onto zeolite Immobilized 100% higher L. productivity rhamnosus ATCC 17% higher yield Fermentation mode Lactic acid 10 concentration (g/l) 0 Lactic acid yield (g/g) Yield coefficient (g/g) Volumetric productivity (g/lh) Immobilized L Time (h) 40 rhamnosus ATCC 7469 Free L. rhamnosus ATCC free L. rhamnosus ATCC Time (h) 0

9 Log N (logcfu g -1 ) Zeolite as a carrier for lactic acid bacteria in biorefinery processes Repeated batch fermentation with L. rhamnosus immobilized onto zeolite After 120h of fermentation number of viable cells has been 9, CFU g -1! Further colonization of zeolite surface during the fermentation High energy was required for detachment of cells strong attachment Time (h) Exopollysacharides Alkaline structure Large surface area Interaction: Zeolite surface Lewis acid L. rhamnosus surface Lewis bases

10 Conclusions Due to the effective immobilization of L. rhamnosus ATCC 7469 onto the zeolite and further colonization of surface during the lactic acid fermentation on stillage, very high number of cells of over CFU g -1 was attached to the surface of zeolite carrier after four subsequent recirculations. Simplicity of the method for immobilization of L. rhamnosus onto zeolite and high lactic acid productivity obtained in the process qualify zeolite as a new and suitable carrier for lactic acid bacteria in biorefinery processes.

11 Acknowledgement This work was funded by Ministry of education, science and technological development, Republic of Serbia, Project number TR Thank you for attention! Faculty of Technology and Metallurgy, University of Belgrade,