The microbial ecology of a rum production process Victoria Green A thesis submitted as a fulfilment for the degree of Doctor of Philosophy University of New South Wales School of Chemical Sciences and Engineering Sydney, Australia March 2015 This thesis is, lovingly, dedicated to Graham Harold Fleet “Fluffy” Supervisor Mentor Friend PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Green First name: Victoria Other name/s: Irene Abbreviation for degree as given in the University calendar: PhD School: Chemical Engineering Faculty: Engineering Title: The microbial ecology of a rum production process Abstract 350 words maximum: (PLEASE TYPE) Rum is an alcoholic beverage made from the distillate of a microbial fermentation of sugar cane molasses. Although commercial rum production started in the 16th century, the microbial ecology of the process has remained relatively unexplored. This thesis reports an investigation of the microorganisms associated with rum production at a distillery in Queensland, Australia. Samples of raw materials (molasses, dunder, water, additives), starter cultures and fermenting molasses were systematically examined for the populations and species of yeasts and bacteria. Molasses contained low populations(< 102 CFU/mL) of yeasts, Bacillus species and lactic acid bacteria. Saccharomyces cerevisiae, used as a starter culture, was the main yeast of molasses fermentation, growing to populations of about 107 CFU/mL. Lactic acid bacteria were consistently isolated from the molasses fermentation and reached populations of 107 CFU/mL. The main species isolated were Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis and an unidentifiable Lactobacillus spp. These species were indigenous contaminants within the processing environment, colon ising sites that escaped effective cleaning and sanitation operations. Species of Clostridium, Zymomonas and Propionibacterium were not detected in the production system. Dunder, which originated from the distillation operation, was considered to be sterile, but developed a population of lactic acid bacteria (the unidentifiable Lactobacillus spp.) on storage. Dunder had significant concentrations of organic acids and amino acids. At concentrations of 10% and above, it significantly inhibited the growth of S. cerevisiae in molasses medium and to a lesser extent lactic acid bacteria. Laboratory scale molasses fermentations and distillations were performed to investigate the effect of lactic acid bacteria on the growth of S. cerevisiae, process efficiency and production of flavour volatiles. Both single and mixed cultures using S. cerevisiae, L. fermentum, L. plantarum and Lactobacillus spp. were undertaken. The bacteria did not restrict the growth of S. cerevisiae but enhanced utilization of molasses sugars and ethanol production. The work presented in this thesis is the first comprehensive and systematic study, of its type, into the microbial ecology of a rum distillery. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). Signature Witness The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS ORIGINALITY STATEMENT 'I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.' Signed Date ii ACKNOWLEDGEMENTS First, and foremost, I would like to extend my gratitude and sincere appreciation to my supervisor, Professor Graham Fleet. He has generously given his guidance, expertise, time and mentorship consistently over the last 9 years. I would also like to acknowledge my co-supervisor Dr Graeme Bushell for his advice and technical expertise. I am very grateful to the Australian Research Council (ARC) and The Bundaberg Distilling Company for funding this research. The technical staff of the Department of Food Sciences and Technology, specifically Camillo Taraborrelli, for his assistance, support and friendship. My thanks to Paul and Phil in the workshop for assisting me with my construction requirements. The administration staff, particularly Ling, Ann, Sandra, John and Rahul, of the School Chemical Engineering for their help and assistance over the years. Undertaking a PhD, both performing the laboratory work, and writing up, can be an isolating, exhausting and mind-numbing experience. I am extraordinarily fortunate to have had many people to lean on during the last 9 years. My fellow PhD students, both from UNSW and other universities, gave me the knowledge that anything and everything is an acceptable feeling during the course of a PhD. From feelings of excitement and motivation to those of fatigue, hopelessness and near surrender, having those people, who have experienced it, by your side is priceless. To my PhD friends, Hugh, Jeremy, James, Matthew, Arjuna, Ai Lin and Alex, thank you for keeping me sane, giving me your advice and encouragement. To my surrogate Sydney family, the UNSW Hockey Club, thank you for being there. I left my home, knowing no-one in Sydney. I left Sydney feeling as though I had lived there for most of my life. Whether it was training, playing, coaching or socialising, there is definitely something special about the Black and Gold. My thanks to everyone I’ve worked with over the years while juggling part time/full time work and study. Thank you for your patience and understanding, your support and your laughter. I’ve had the pleasure of working with some amazing people at 3M and ACTGAL. iii Heartfelt thanks to my family, extended family and friends. I am grateful for the encouragement, friendship, care and love you have all shown me over the years. I treasure our times together and look forward to many more now that I have, those things called, weekends back! My dearest friend Elmo, no matter where you have been on this Earth physically, you’ve always been next to me in spirit. Thank you for having faith. To my husband, Matt; your support and love through all that has happened these last 7 years knows no limits. Words cannot express my thanks. Finally, Mum and Dad, thank you for your continued love. Mum, your letters were a great comfort when I moved to Sydney. Dad, who would have thought reading the newspaper would lead to this? Victoria Green (2015) iv LIST OF TABLES AND FIGURES Title Page Number Table 2.1 Rum production statistics for 2011 by leading brand names 14 Figure 2.1 Outline of the process of rum production from molasses 15 Table 2.2 Chemical composition of blackstrap molasses 18 Table 2.3 Dunder definitions used for rum production 23 Table 2.4 Chemical properties of dunder (distillery wastewater, 25 molasses fermentations) Figure 2.2 Pot stills used in the production of distilled spirits 32 Figure 2.3 Distillation at the Bundaberg Distilling Company 34 Figure 2.4 Interactions occurring during maturation of raw rum spirits in 36 an oak barrel Table 2.5 HACCP principles 40 Table 2.6 Important flavour volatiles in rum 45 Table 2.7 Microorganisms found in raw materials, such as molasses 48 and sugar cane juice, associated with alcoholic fermentations for rum production Table 2.8 Microbial species associated with the fermentation of 52 molasses or sugar cane juice for rum or rum agricole production Table 2.9 Properties required by rum yeasts to be commercially 59 successful as a starter culture Table 3.1 Sampling sites for microbiological study of a rum distillery 66 Figure 3.1 Flow diagram of rum production process showing sites of 72 sampling for microbiological analysis as given in Table 3.1 Figure 3.2 Process steps at the Bundaberg Distilling Company 73 Figure 3.3 Raw materials used in the production of rum 75 Figure 3.4 Yeast propagation steps undertake at the Bundaberg distillery 76 Table 3.2 Morphology and sequence identification of yeasts associated 81 with rum production Figure 3.5 Scanning electron microscope image of Saccharomyces 82