**1. Introduction**

Over the years, climate changes and air pollution attributed to the use of fossil fuels has been an increased concern all around the world [1]. One of the main reasons related to the actual environmental issues is the high amount of greenhouse gases (GHG) continuously released into the atmosphere due to anthropogenic activities (industries, land use, transportation, etc.) [2]. In this context, the use of biofuels such as bioethanol has emerged as a sustainable alternative to fossil fuels due to numerous advantages, including reduced emission of GHG, hydrocarbons and nitrogen oxides during both bioethanol production and combustion [3]. In this context, many countries have been adopting new policies to promote the use of biofuels, and the market perspectives are a 2-fold increase in Brazil and the USA and a 4-fold increase in China and EU by 2040 [4].

Among biofuels, bioethanol production through anaerobic fermentation of carbohydrates by yeast microorganisms is the most developed and implemented process at an industrial scale [5]. Fermentable sugars are generally extracted from various plants such as corn, sugarcane and sugar beet, which represent the main feedstock used for the actual worldwide ethanol production [6]. However, even if part of the carbon released after the fuel combustion is captured by the crops during their growth, the production of bioethanol from conventional crops can still impact carbon neutrality [4].

**Citation:** Beigbeder, J.-B.; de Medeiros Dantas, J.M.; Lavoie, J.-M. Optimization of Yeast, Sugar and Nutrient Concentrations for High Ethanol Production Rate Using Industrial Sugar Beet Molasses and Response Surface Methodology. *Fermentation* **2021**, *7*, 86. https:// doi.org/10.3390/fermentation 7020086

Academic Editors: Giuseppa Di Bella and Alessia Tropea

Received: 5 May 2021 Accepted: 25 May 2021 Published: 31 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

One way to reach a carbon neutrality (or even negative carbon emissions) could involve reusing biomass residues such as agricultural and food wastes as alternative feedstocks [7]. These by-products represent a concrete solution to avoid the direct land use impact of using crops for the production of fuels, especially in light of life cycle assessment calculations that will determine the carbon impact of biofuels [8]. Recent studies reported the use of various residues such as food waste [9], corncobs [10] and algae waste [11] for the sustainable production of bioethanol. In the case of lignocellulosic biomass, several pre-treatment steps are often required to break down the complex lignin structure and depolymerise the crystalline cellulose to access fermentable sugars [12]. However, residues from the food industry often have the advantage of being rich in easily fermentable carbohydrates, which could simplify the overall process while reducing the energy requirements for the process and possibly, the carbon intensity of the downstream fuel [13].

Sugar beet and cane molasses are abundant liquid by-products from the sugar industry, which are generally found at high amount of total sugars (50.6–71.0% *w*/*w*) and traces of micronutrients such as minerals (Ca, Mg, Na and K), phosphate and nitrogen compounds [14]. These sugar-rich solutions does not require any major physical or chemical pretreatment (such as hydrolysis, filtration, sterilization, etc.) before fermentation, making them very appropriate for ethanol production. In this context, several studies already reported the efficient production of bioethanol using sugar beet and sugarcane molasses. For instance, Razmovski et al. compared the fermentation performances of immobilised and free yeast cells using sugar beet molasses and thick juice at three initial glucose concentrations of 100, 200 and 300 g/L [13]. For both substrates, immobilised yeasts resulted in higher ethanol yields than free cells condition, with the highest ethanol volumetric productivity of 1.257 and 1.422 g/L/h obtained with molasses and thick juice diluted at 150 g/L of initial sugar, respectively. In another study, the continuous production of ethanol was investigated using an immobilised yeast cell reactor and sugarcane molasses as low-cost fermentation substrate [15]. The highest ethanol production of 19.15 g/L was obtained with a hydraulic retention time of 15.63 h combined with an initial sugar concentration of 150 g/L. In a recent study, a rotary biofilm reactor was developed for long-term bioethanol production using non-sterilised sugar beet molasses [16]. By recycling 30% of the fermentation broth every 36 h, a stable production of 52.3 g/L of ethanol was achieved over a period of 60 days. In addition, molasses can also be used for the production of different alcoholic beverages such as rum, a spirit distillate with an ethanol content of 37–43% alcohol by volume [17]. In another study, the ethanol production performances of corn marsh feedstock were improved by the addition of sugarcane molasses [18]. Mixing 50% of corn mash with 50% of sugar cane molasses generated the highest ethanol concentration (8.2%).

To implement an efficient fermentation process using industrial by-products, several process parameters such as sugar and yeast concentrations must be optimised to ensure high ethanol productivity while keeping in mind the economic aspects of the process [19]. Nutrient supplementation is also an important parameter to take into consideration since an adequate amount of nutrient can significantly improve yeast viability and resistance to the medium, stimulating ethanol production performances. Since alcoholic fermentation is a complex biological process involving various operating factors, the use of the classical "one factor at a time" approach could be time-consuming due to the large number of experiments to perform. Hence, tools such as the statistical design of experiment (DoE) allow investigating the effect of several operational factors as well as their interactions on the overall process while considerably reducing the number of experimental tests [20]. For instance, a central composite design coupled with response surface methodology (CCD-RSM) represents a powerful and effective statistical tool that could commonly be used for the optimization of biotechnological processes such as fermentation [19,21,22]. Once developed, the CCD-RSM can be used to predict a process output, while imposing specific constraints based on economical and technical aspects.

In this context, the present work aims to optimise the production of ethanol from non-treated sugar beet molasses produced by a local sugar refinery. For this purpose, a CCD-RSM was designed and developed to investigate the effect of three fermentation process parameters (initial sugar, yeast and nutrient concentrations) on ethanol productivity while considering several operating parameters such as ethanol yield and sugar utilization rate. Then, the second-order mathematical model obtained through the CCD-RSM was tested to evaluate its ability to make accurate predictions based on specific desired process outputs. To the best of our knowledge, this is the first study reporting the use of a CCD-RSM statistical approach to maximise the production of ethanol from non-sterilised sugar beet molasses while scaling up the experimental results up to a 100 L bioreactor scale.
