_____________________________________________________________________________________________________ *Corresponding author: E-mail: nkengozi202@yahoo.com; Journal of Advances in Microbiology 21(4): 17-33, 2021; Article no.JAMB.66414 ISSN: 2456-7116 Bioethanol Production from an Underutilized Plant, Calabash (Crescentia Cujete) Using Co-Culture of Saccharomyces Cerevisiae and Cronobacter Malonaticus N. U. Nwogwugwu 1,2* , G.O. Abu 2,3 and O. Akaranta 2,4 1 Department of Microbiology, Federal University of Technology, Owerri, Nigeria. 2 Centre for Oil Field Chemicals Research, University of Port Harcourt, Nigeria. 3 Department of Microbiology, University of Port Harcourt, Port Harcourt, Nigeria. 4 Department of Pure and Applied Chemistry, University of Port Harcourt, Nigeria. Authors’ contributions This work was carried out in collaboration among all authors. All authors read and approved the final manuscript. Article Information DOI: 10.9734/JAMB/2021/v21i430339 Editor(s): (1) Dr. Foluso O. Osunsanmi, University of Zululand, South Africa. Reviewers: (1) Gabriel Sanjo Aruwajoye, University of KwaZulu-Natal, South Africa. (2) Alaika Kassim, University of KwaZulu-Natal (UKZN), South Africa. (3) Carolina Brito Codato Zumpano, University of São Paulo, Brazil. Complete Peer review History: http://www.sdiarticle4.com/review-history/66414 Received 12 January 2021 Accepted 19 March 2021 Published 17 April 2021 ABSTRACT Response surface methodology (RSM) model was used to optimize ethanol production from calabash (Crescentia cujete) pulp juice using co-culture of Saccharomyces cerevisiae and Cronobacter malonaticus. The calabash pulp was squeezed with muslin cloth, and vacuum filtered to clear solution before use. The clear juice was tested for reducing sugars using the Dinitrosalicylic acid (DNS) method. Twenty three runs (23), including 3 controls, of the fermentation were conducted at varying temperatures, pH, and volumes of inoculum. The process parameters (input variables): volumes of inoculum, temperature, and pH were subjected to response surface model, using the Central composite design (CCD). Fermentation was done in conical flasks covered with cotton wool and foil in a stationary incubator for four days (96 hours). Active co-culture of Saccharomyces cerevisiae and Cronobacter malonaticus was used, with inoculum developed using Original Research Article