Growth of Methylobacterium organophilum in Methanol for the Simultaneous Production of Single-Cell Protein and Metabolites of Interest

Abstract

Research background. This study aims to monitor the growth of the methylotrophic bacteria Methylobacterium organophilum in a culture medium with methanol as a carbon source and to verify the production of unicellular proteins and other biomolecules, such as carotenoids, exopolysaccharides and polyhydroxyalkanoates, making them more attractive as animal feed. Experimental approach. Bacterial growth was studied in shake flasks using different carbon/nitrogen (C:N) ratios to determine their best ratio for achieving the highest volumetric productivity of cells and substrate consumption rate. This optimal parameter was further used in a fed-batch operating bioreactor system to define the kinetic profile of cell growth. Methanol consumption was measured by HPLC analysis and the extracted pigments were analyzed by liquid chromatography/mass spectrometry. Chemical composition and rheological properties of the produced exopolysaccharides were also determined. Results and conclusions. The best experimental parameters were verified using an initial methanol concentration of 7 g/L in the culture medium. The same initial substrate concentration was used in the fed-batch operation and after 60 h of cultivation 5 g/L of biomass were obtained. The accumulation of carotenoids associated with cell growth was monitored, reaching a concentration of 1.6 mg/L at the end of the process. These pigments were then analyzed and characterized as a set of xanthophylls (oxidized carotenoids). In addition, two other product types were identified during the fed-batch operation: exopolysaccharides, which reached a concentration of 8.9 g/L at the end of the cultivation, and an intracellular granular structure that was detected by transmission electron microscopy (TEM), suggesting the accumulation of polyhydroxyalkanoate (PHA), most likely polyhydroxybutyrate. Novelty and scientific contribution. Methylobacterium organophilum demonstrated a unique ability to produce compounds of commercial interest. The distinct metabolic diversity of this bacterium makes room for its use in biorefineries.

Alternate abstract:

Pozadina istraživanja. Svrha je ovoga istraživanja bila pratiti rast metilotrofne bakterije Methylobacterium organophilum u podlozi s metanolom kao izvorom ugljika, te potvrditi sposobnost tog jednostaničnog organizma da proizvodi proteine i druge biomolekule, kao što su karotenoidi, egzopolisaharidi i polihidroksialkanoati, a koji se mogu upotrijebiti u proizvodnji stočne hrane. Eksperimentalni pristup. Rast bakterije ispitan je na tresilici pri različitim omjerima ugljika i dušika (C:N), radi utvrđivanja njihovog optimalnog omjera za postizanje najveće volumetrijske produktivnosti stanica i najboljeg iskorištenja supstrata. Zatim je pomoću tog optimalnog omjera hranjiva određen kinetički profil rasta stanica u šaržnom bioreaktoru. Potrošnja metanola mjerena je metodom HPLC, a izolirani su pigmenti ispitani tekućinskom kromatografijom spregnutom s masenom spektrometrijom. Također su određeni kemijski sastav i reološka svojstva dobivenih egzopolisaharida. Rezultati i zaključci. Najbolji eksperimentalni parametri potvrđeni su pri početnoj koncentraciji metanola u podlozi od 7 g/L. Ista je početna koncentracija korištena u šaržnom bioreaktoru, gdje je nakon 60 h uzgoja dobiveno 5 g/L biomase. Tijekom uzgoja je praćena akumulacija karotenoida, čija je koncentracija pri završetku procesa bila 1,6 mg/L. Pigmenti su zatim ispitani i okarakterizirani kao ksantofili (podvrsta karotenoida). Osim toga, tijekom šaržne fermentacije identificirana su još dva proizvoda: egzopolisaharidi, čija je koncentracija na kraju uzgoja bila 8,9 g/L; te granularna struktura otkrivena pomoću transmisijskog elektronskog mikroskopa, koja upućuje na to da je u stanici došlo do nakupljanja polihidroksialkanoata (PHA), i to najvjerojatnije polihidroksibutirata. Novina i znanstveni doprinos. Bakterija Methylobacterium organophilum ima jedinstveno svojstvo proizvodnje spojeva od komercijalnog interesa. Iznimna metabolička raznolikost ove bakterije omogućuje njezinu primjenu u biorafinerijama.