Bioresource Technology 340 (2021) 125595 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech A preliminary study on a novel bioaugmentation technique enhancing lactic acid production by mixed cultures fermentation Grazia Policastro a,*, Federica Carraturo b, Mariacristina Compagnone c, Marco Giugliano a, Marco Guida b, Vincenzo Luongo d, Raffaele Napolitano a, Massimiliano Fabbricino a a Department of Civil, Architectural and Environmental Engineering, University of Naples Federico II, via Claudio 21, Naples 80125, Italy b Department of Biology, University of Naples Federico II, via Cintia 21, Naples 80126, Italy c Department of Chemical Engineering and Food Technologies, Faculty of Sciences, University of Cadiz,´ 11510 Puerto Real, Cadiz,´ Spain d Department of Mathematics and Applications Renato Caccioppoli, University of Naples Federico II, via Cintia, Monte S. Angelo, Naples I-80126, Italy HIGHLIGHTS GRAPHICAL ABSTRACT • A bioaugmentation technique to select cultures producing lactic acid is proposed. • The selection occurs via sequential batch steps with intermittent pH correction. • Substrate concentration affects the effi­ cacy of the bioaugmentation technique. • As consequence of the bioaugmentation, lactic acid increases from 5% to 59% • The technique results in microbial pop­ ulation change to Bacillus species. ARTICLE INFO ABSTRACT Keywords: The paper is a preliminary study on the selection of lactic acid producing microorganisms from a mixed microbial Bacilli population via bioaugmentation. The bioaugmentation technique is based on pH sudden variations occurring in Fermentation sequential batch steps of a dark fermentation process applied to simple substrates. Different conditions are tested Lactic acid and compared. The structure of microbial communities and concentrations of metabolic intermediates are Microbial selection analyzed to study the possible substrate conversion routes. Obtained results indicate that the initial mixed culture Mixed cultures produced a lactic acid percentage of 5% in terms of CODLA/CODPRODUCTS. In the most favourable conditions, the selected culture produced a lactic acid percentage of 59%. The analysis of the composition of microbial com­ munities before and after the bioaugmentation processes, indicates that lactic acid production mainly results from the population change to bacteria belonging to the genus Bacillus. Indeed, the relative abundance of Bacilli increased from 0.67%, to 8.40% during the bioaugmentation cycle. * Corresponding author. E-mail addresses: [email protected] (G. Policastro), [email protected] (F. Carraturo), [email protected] (M. Compagnone), [email protected] (M. Giugliano), [email protected] (M. Guida), [email protected] (V. Luongo), raffaele.napolitano13@studenti. unina.it (R. Napolitano), [email protected] (M. Fabbricino). https://doi.org/10.1016/j.biortech.2021.125595 Received 10 June 2021; Received in revised form 13 July 2021; Accepted 14 July 2021 Available online 26 July 2021 0960-8524/© 2021 Elsevier Ltd. All rights reserved. G. Policastro et al. Bioresource Technology 340 (2021) 125595 1. Introduction Table 1 Operative conditions of fermentative steps (IiSj). The development of new strategies for an efficient production of Cycle Step Step Step F/M ratio Substrate lactic acid (LA) is of great interest, due to the wide application of this # 1 2 3 (mgCOD/mgCOD) concentration (g/L) product in chemical, pharmaceutical and food industries (Luongo et al., 1 I1S1 I2S1 I3S1 0.025 0.60 2019). Moreover, LA can be used to produce a polymer (polylactic acid) 2 I1S2 I2S2 I3S2 0.5 1.20 able to replace petrochemical plastics in several applications (Liang 3 I1S3 I2S3 I3S3 0.1 2.40 et al., 2015). This is why LA market is supposed to reach 9.8 billion USD 4 I1S4 I2S4 I3S4 0.25 6.01 5 I S I S I S 0.5 12.03 by 2025 (Rombouts et al., 2020), with an annual growth rate of almost 1 5 2 5 3 5 19% (Oliveira et al., 2020). Among different LA production alternatives, microbial fermentation bioaugmentation cycle, is based on the development of three sequential is gaining increasing attention, being the less expensive and the most fermentative steps, conducted in batch conditions. The selection of LAB environmental friendly solution (Rawoof et al., 2021). simply occurs as consequence of pH variations, induced by the applied Currently, pure bacterial fermentations account for 90% of LA pro­ organic charge and by the punctual pH corrections effectuated at the duction worldwide (Oliveira et al., 2020), although the use of mixed beginning of each step. To optimize the bioaugmentation efficiency, cultures would be more attractive, because of its intrinsic economical various Food to Microorganisms (F/M) ratios are tested. No nutrient and operational advantages, including the possibility of using waste addition is operated, as the study is aimed at developing a technique material as bacterial substrates, with no need of sterilisation (Regueira which could be applied in the presence of simple substrates. Therefore, et al., 2021). Indeed, the use of mixed culture fermentation for LA the present study represents an indication to address future researches production is still challenging to date. Recently, it has been reported that aimed at producing lactic acid from both unrelated substrates and LA may result as the dominant product of mixed microbial fermentations external inocula, acting on operational conditions only. if specific substrates are used. Rombouts et al., (2020) studying mixed fermentation for LA production, observed that the microbial community 2. Materials and methods could be dominated by lactic acid bacteria (LAB) (e.g. Lactococcus and Lactobacillus species) using a rich medium containing peptides and B 2.1. Experimental apparatus and materials vitamins. On the contrary, employing a simple mineral medium, the substrate is mainly fermented to acetate and butyrate by species Experimental tests were conducted in batch mode, at mesophilic ◦ belonging to the Clostridia class. temperature (35 ± 1 C), using 500 mL glass reactors (400 mL working Luongo et al., (2019) observed that indigenous cultures of cheese volume). The reactors were equipped with two different ports, used for whey allow obtaining LA as the main fermentation product. At the same liquid and gas sampling operations. Plastic tubes and gaskets were used time, the authors proved that the use of an external inoculum for LA for junctions. Before use, sealing joints were controlled, filling each production from cheese whey at uncontrolled pH, resulted in acetic acid reactor with water and pressurized air. High purity chemicals were used and ethanol production. The study also demonstrated that LAB are able for substrate preparation and all analytical determinations. Adopted to grow at extremely low pH (Luongo et al., 2019). The external pH glassware were soaked overnight in a nitric acid bath (2% v/v) and decrease do not constitute an acid shock as LAB are naturally able to rinsed several times with bi-distilled water. acidify the external medium (Siegumfeldt et al., 2000). Moreover, LAB are more acid-tolerant than other fermentative bacteria (Padmavathi et al., 2018; Wang et al., 2018). Consequently, various researchers 2.2. Bioaugmentation cycle adopted acidic pre-treatments to favour LAB proliferation in mixed culture fermentation. This procedure limited the production of other The bioaugmentation cycle was composed by three successive catabolites, and increased the lactic acid percentage in the fermentation fermentative steps, each conducted using, as inoculum (Ii=1-3), the broth (Choi et al., 2016; Itoh et al., 2012). biomass selected in the previous step. The external mixed culture, used Generally, a rapid change of the external pH represents a stressful as initial inoculum (I1), was sampled from a full-scale anaerobic treat­ situation for microorganisms. However, differently from most non-lactic ment plant located in Casal di Principe (South of Italy), processing the acids producing bacteria, LAB are able to regulate their intercellular pH organic fraction of municipal solid waste. The characteristic of the as an adaptation strategy (Siegumfeldt et al., 2000). When the external digestate were: COD = 77.30 ± 0.08 g/L, pH = 8. Before use, the pH decrease, neutrophilic bacteria maintain an internal pH that is close inoculum was pre-treated by heat shock, as detailed described elsewhere to 7. The high internal-external pH difference generates large proton (Luongo et al., 2019). Used substrate for each fermentation step was a gradients, which is disadvantageous for fermentative bacteria. Indeed, synthetic glucose solution, prepared dissolving the solid compound in proton translocation requires energy (Kobayashi et al., 1986). On the bi-distilled water. other hand, many acid-tolerant microorganisms, such as LAB, have Five different operative conditions were tested, corresponding to five developed a different strategy: The internal pH decreases as the external different initial concentrations of the substrate (Sj = 1–5), as summarized pH decreases to maintain a constant pH gradient rather than a constant in Table 1. Substrate concentrations were calculated setting different internal pH (Siegumfeldt et al., 2000). Several possible methods which initial F/M ratios. Each condition was conducted in triplicates. For can be used by bacteria to regulate the internal pH have been studied,