Agroindustrial by-products and residues can be transformed into valuable compounds in 24 biorefineries. Here we present a new concept: production of fuel ethanol, whey protein and 25 probiotic yeast from cheese whey. An initial screening under industrially relevant conditions, 26 involving thirty Kluyveromyces marxianus strains, was carried out using spot assays to 27 evaluate their capacity to grow on cheese whey or on whey permeate (100 g lactose/L), 28 under aerobic or anaerobic conditions, in the absence or presence of 5% ethanol, at pH 5.8 or 29 pH 2.5. The four best growing K. marxianus strains were selected and further evaluated in a 30 miniaturized industrial fermentation process using reconstituted whey permeate (100 g 31 lactose/L) with cell recycling (involving sulfuric acid treatment). After five consecutive 32 fermentation cycles, the ethanol yield on sugar reached 90% of the theoretical maximum in 33 the best cases, with 90% cell viability. Cells harvested at this point displayed probiotic 34 properties such as capacity to survive the passage through the gastrointestinal tract and 35 capacity to modulate innate immune response of intestinal epithelium, both in vitro. 36 Furthermore, the CIDCA 9121 strain was able to protect against histopathological damage in 37 an animal model of acute colitis. Our findings demonstrate that K. marxianus CIDCA 9121 38 is capable of efficiently fermenting the lactose present in whey permeate to ethanol and that 39 the remaining yeast biomass has probiotic properties, enabling an integrated process for the 40 obtainment of whey protein, fuel ethanol and probiotics from cheese whey. 41 42 2. Importance 43 Cheese whey is the liquid remaining following the precipitation and removal of milk casein 44 during cheese-making. This by-product represents about 85-95% of the milk volume and 45 retains 55% of milk nutrients so it can be exploited as a source of valuable end products. 46 However, at a global level around 50% of cheese whey is wasted, representing an important 47 environmental impact and indicating the need to develop alternative processes to recover 48 value. Kluyveromyces marxianus is capable of fermenting lactose, generally regarded as safe, 49 and has been explored separately as an ethanol producer and as a viable bioactive 50 microorganism. The significance of our research is to establish the proof of concept that a 51 biorefinery for fuel ethanol production using whey and K. marxianus can also be exploited to 52 obtain viable probiotic biomass, conferring an added value to the process and providing an 53 alternative to reduce environmental impact. 54 55 3
Introduction
56Whey production around the world is over 160 million tons per year and it is continuously 57 increasing (1). Whey is characterized by high ranges of biochemical oxygen demand (BOD) 58 and chemical oxygen demand (COD), 30-50 g/L and 60-80 g/L, respectively, mainly due to 59 its lactose content. In recent years, there has been a significant increase in alternative 60 p...