Citation: Dmitrovi´ c, S.; Luki´ c, N.; Pajˇ cin, I.; Vlajkov, V.; Grahovac, J.; Joki´ c, A. The Use of Chitosan for Flocculation Recovery of Bacillus Biomass Grown on Dairy and Wine Industry Effluents. Processes 2023, 11, 1099. https://doi.org/10.3390/ pr11041099 Academic Editors: Bruna De Falco and Konstantina Tsikrika Received: 20 March 2023 Revised: 28 March 2023 Accepted: 3 April 2023 Published: 4 April 2023 Copyright: © 2023 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/). processes Article The Use of Chitosan for Flocculation Recovery of Bacillus Biomass Grown on Dairy and Wine Industry Effluents Selena Dmitrovi´ c *, Nataša Luki´ c, Ivana Pajˇ cin , Vanja Vlajkov , Jovana Grahovac * and Aleksandar Joki´ c Faculty of Technology Novi Sad, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia; nlukic@tf.uns.ac.rs (N.L.); ivana.pajcin@uns.ac.rs (I.P.); vanja.vlajkov@uns.ac.rs (V.V.); jokic@uns.ac.rs (A.J.) * Correspondence: selena.dmitrovic@uns.ac.rs (S.D.); johana@uns.ac.rs (J.G.) Abstract: The downstream processing of efficient biomass-based microbial biopesticides is heavily reliant on obtaining the largest concentration of viable cells in the most cost-effective manner. The goal of this research was to assess the ability of chitosan flocculation to recover bacterial Bacillus sp. BioSol021 biomass from the broth after biological treatment of wastewaters from the dairy and wine industries. Second-order factorial design models were used to estimate the effect of chitosan concentration and mixing speed on flocculation efficiency, settling velocity, and antimicrobial activity against Aspergillus flavus, i.e., inhibition zone diameter. Response surface methodology was followed by multi-objective optimization by applying the desirability function (DF) and genetic algorithm (GA). The optimum values for flocculation efficiency, settling velocity, and inhibition zone diameter for cheese whey effluent were 88%, 0.10 mm/s, and 51.00 mm, respectively. In the case of winery flotation effluent, the optimum values were flocculation efficiency 95% and settling velocity 0.05 mm/s, while the inhibition zone diameter was 48.00 mm. These results indicate that utilizing chitosan as a flocculation agent not only fits the criteria for effective downstream processing, but also has a synergistic effect on Bacillus sp. antibacterial activity. Keywords: Bacillus sp.; flocculation; chitosan; food industry wastewaters 1. Introduction Food spoilage organisms are widely regarded as a severe public health threat and provide significant health concerns to consumers and the food industry [1,2]. The use of natural antimicrobial agents represents a highly effective approach for biopreservation of food products and a significant part of the green food revolution. Some plant pathogens, such as fungi Aspergillus flavus, beside the problem of food spoilage, release mycotoxin contamination that could influence human health. A well-known opportunistic pathogen of numerous crops with economic value, such as maize, peanuts, and cotton, Aspergillus flavus is also a noteworthy producer of aflatoxins [3,4]. Aflatoxin B1 (AFB1) is categorized by the International Agency for Research on Cancer (IARC) in the 1a group of substances that are carcinogenic to both humans and animals. AFB1 is one of the few mycotoxins that have been used in the development of biological weapons and is regarded as the most potent natural carcinogen [5,6]. Plant growth promotion, induction of systemic plant resistance, biofilm formation, competition for nutrients and space, lytic effect, and antibiotic production are just a few of the diverse mechanisms of action used by various bacterial biopesticides of the Bacillus genus whose cells and active metabolite components display intense antibacterial and antifungal activity against the different plant pathogens [7]. The substitution of chemical pesticides by biological ones is a foreseeable trend. However, a number of obstacles have prevented the commercial production and use of microbial products. Despite the substantial potential of currently known and utilized biopesticides, there are still only a few products on the market at large. The high cost of biotechnological production, with a focus on the Processes 2023, 11, 1099. https://doi.org/10.3390/pr11041099 https://www.mdpi.com/journal/processes