Bioaugmentation with Enterococcus casseliflavus: A Hydrogen-Producing Strain Isolated from Citrus Peel Waste



To isolate, identify and characterize an anaerobic strain from in natura CPW, in relation to the consumption of different carbon sources, growth kinetics and H2 production after bioaugmentation.


Several substrates were evaluated (glucose, fructose, sucrose, xylose, starch, cellobiose, cellulose and lactose), and its optimal concentration was evaluated by Experimental Design method, besides the application of the isolated strain in citrus peel waste (CPW).


A facultative anaerobic strain, similar (99%) to Enterococcus casseliflavus, was isolated from in natura citrus pulp and bagasse. Xylose was the monomer from which there was greater H2 production (10.3 mmol L−1). For 0.5 to 6.5 gxylose L−1, 3.0 to 12.9 mmol H2 L−1 was obtained, respectively. Generation time (Tg) of 0.35 h and specific growth (µ) of 1.98 h−1 for pH 7.0, 37 °C and 2 g glucose L−1 was obtained for E. casseliflavus. In bioaugmentation assays (CPW + E. casseliflavus), highest maximum H2 production potential (P = 13.9 mmol L−1), maximum H2 production rate (Rm = 1.09 mmol h−1) and time to start the H2 production (λ = 2.12 h−1) was observed when compared to glucose (P = 9.1 mmol L−1; Rm = 1.99 mmol h−1; λ = 4.08 h−1).


From glucose, there was prevalence of butyric acid pathway (759 mg L−1) in relation to acetic acid (303 mg L−1). From CPW, there was higher production of acetic acid (878 mg L−1) in relation to butyric (147 mg L−1). The application of E. casseliflavus by bioaugmentation is an alternative for biodigestion of complex substrates, such as CPW, to obtain products of biotechnological interest.

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The authors would like to thank the technical support of Dr. Maria Angela Adorno and Dr. Carolina Sabatini. This work was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (processes 2017/01722-0 and 2015/06246-7) and was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001.

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Camargo, F.P., Sakamoto, I.K., Silva, E.L. et al. Bioaugmentation with Enterococcus casseliflavus: A Hydrogen-Producing Strain Isolated from Citrus Peel Waste. Waste Biomass Valor 12, 895–911 (2021).

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  • Lignocellulosic biomass
  • Autochthonous consortia
  • Agro-industrial waste
  • Nutritional evaluation