Microsoft Word - 19Venkatesan.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 80, 2020 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Eliseo Maria Ranzi, Rubens Maciel Filho Copyright © 2020, AIDIC Servizi S.r.l. ISBN 978-88-95608-78-5; ISSN 2283-9216 Second-generation (2G) Lactic Acid Production and New Developments – A Mini-review Regiane Alves de Oliveiraa,c, Carlos E. Vaz Rossellb, Gonçalo A. G. Pereiraa, Rubens Maciel Filhoc,* a Laboratory of Genomics and Bioenergy, Institute of Biology, University of Campinas – Unicamp, Rua Carl Von Linaeus, S/N, 13083-864, Campinas-SP, Brazil b Interdisciplinary Center of Energy Planning, University of Campinas – Unicamp, Rua Cora Coralina, 330, 13083-896, Campinas-SP, Brazil c Laboratory of Optimization, Design and Advanced Process Control, School of Chemical Engineering, University of Campinas – Unicamp, Avenida Albert Einstein, 500, 13083-852, Campinas-SP, Brazil maciel@feq.unicamp.br Lactic acid (LA) production is already a global reality. Its applications cover the most diverse industrial sectors and have rapidly consolidated in recent years. Currently, the most prominent use of LA is the production of polylactic acid to replace plastics from the petrochemical industry. A great part of this rapid change is due to the rising worldwide concerns about the excess of non-degradable plastics used daily and the accumulation of this material in nature. In this scenario, LA production becomes even more relevant when considering its production from renewable raw materials, especially second-generation (2G) substrates, such as lignocellulosic biomass. This reduces the human dependence on oil for both energy and fuel production, as well as for the production of plastics and other chemicals since LA is still one of the most relevant building block chemicals. Nowadays it is possible to produce LA from the most diverse 2G-substrates available around the world. Thus, LA production by fermentation of 2G-sugars can be associated with several existing biorefinery models, such as for biofuels and chemicals production. In this scenario, for example, it is possible to associate LA production with ethanol production in a biorefinery model, producing 1G-ethanol, 2G-LA, sugar for food, and electricity. This kind of approach may represent a break from current production model of energy and chemicals to a more sustainable and democratic scenario, including new players in the world market and reducing the dependence of other countries to supply oil and its derivates, especially when associated with new and powerful genetic engineering tools. Front this scenario, this review presents the state of the art of 2G-LA production. 1. Introduction Lactic acid (LA) is classified as 2-hydroxy-propanoic acid (C3H6O3) and is one of the most relevant carboxylic acids in the world. Several well-known industrial applications include their constant use in the food, pharmaceutical and cosmetics industries (Mazzoli et al., 2014). In addition, its use in the production of polylactic acid (PLA) with broad applications in the substitution of petroleum-derived polymers has sparked worldwide interest in this versatile chemical molecule. Its versatility is mainly due to its natural occurrence in the isomeric forms of L and D LA. Each of the isomers has different applications, being the L-isomer the most used by the food, cosmetic and pharmaceutical industries, due to its long-known compatibility with the human body. On the other hand, different mixing rates of the D and L isomers may confer interesting properties in the construction of LA derived polymers, altering their resistance, flexibility, crystallinity and degradation rate (Mazzoli et al., 2014). Thus, the PLA industry can build polymers that replace almost any known polymer derived from the petrochemical industry, besides producing biodegradable plastics for daily use. Petrochemical LA production generates a racemic LA mixture, that, once polymerized will result in an amorphous PLA (Figure 1a), with a low real use for the industry. The ability to produce a high purity isomer has important ramifications in the chemistry and ultimate process/property relationships achievable in the DOI: 10.3303/CET2080038 Paper Received: 13 November 2019; Revised: 22 January 2020; Accepted: 24 April 2020 Please cite this article as: Alves De Oliveira R., Vaz Rossell C.E., Pereira G.A., Maciel Filho R., 2020, Second-generation (2g) Lactic Acid Production and New Developments – a Mini-review, Chemical Engineering Transactions, 80, 223-228 DOI:10.3303/CET2080038 223 polymers produced from LA. In fact, LA polymers with specific characteristics may be produced to medical applications using controlled amounts of L and D isomers. In this sense, efficient producers of LA microbial species have LA as a single fermentation product from hexoses and pentoses. The fermentation pathways of lactic acid bacteria (LAB) have high conversion rates, which means large amounts of sugar are fermented and not much sugar is converted for cell biomass production. It leads to high yields of LA and makes them industrially interesting (Sauer et al., 2017). A further interesting ability is that many LAB are able to utilize various hexoses and pentoses (Sauer et al., 2017), making them perfect to exploit lignocellulosic sugars to produce second-generation (2G) LA. Figure 1: a) Lactic acid production route from a petrochemical source; and b) Lactic acid production route from an organic source. Thus, considering different sugar release technologies already available, LAB have the potential to convert almost any organic source into isomeric pure LA (Figure 1b). LAB have high sugar uptake rates, which strongly contribute to high productivities and economically benefits the microbial production process of LA production. The high sugar uptake rates are comparable to other largely used microbial biotech microorganisms, such as E. coli or S. cerevisiae. Considering the presented characteristics, the next steps of biotech development related to LAB goes toward the development of genetic toolboxes to make these microorganisms efficient cell factories, not only regarding LA production, but also other industrial molecules (Sauer et al., 2017). LAB are considered fastidious microorganisms, requiring organic sources of nitrogen and vitamins. In some cases, it can be considered a disadvantage for the use of LAB for chemical production. However, the use of 2G substrates has the potential to overcome those needs, since they can be complex organic material instead of pure processed sugars. In this sense, different types of biomass, such as energy crops, forestry residues, or by-products from agroindustry activity, featuring both the low purchase cost and circular economy, have already been tested as fermentative substrates for 2G-LA production. Taking these into account, the main achievements of the 2G-LA research are presented in this short review. 2. Fermentation feedstock for 2G-lactic acid production Nowadays, LA applications are limited due to its high cost of production, mainly because of the cost of carbon source (glucose), showing the importance of exploring alternative cheaper carbon sources for LA production (Jiang et al., 2019). According to Guo et al. (2015), 2G-products are those produced from non-food materials, such as agricultural residues, wood, and energy crops typically high in lignocellulose, among others. Many studies propose 2G-LA production as a viable scenario, showing that besides the numerous obstacles to be overcome, this technology presents many advantages. Considering this definition of 2G-products, some recent studies of 2G-LA production are presented in Table 1. Considering the data, it is clear that the viability of 2G-LA production broadly varies according to the combination of several factors, such as substrate, microbial strain, and operational conditions. Besides, it is also important to consider the stable availability of the substrates over the year, as well as its transportation, storage, and logistics, in order to make the process economically feasible and sustainable. Once considered these factors, an important advantage of using those substrates is its availability in several regions of the globe, which makes it possible to produce LA (and other bulk chemicals) in a great diversity of places around the world. Finally, some of the main challenges in LA production using 2G-substrates can be solved by new genetic engineering tools in development for LAB, such as substrate recalcitrance, carbon catabolite repression, by- product formation, optical purity, LA and pH inhibition, sensibility to inhibitors released in the biomass pretreatment (Cubas-Cano et al., 2018; Upadhyaya et al., 2014). 224 Table 1: Second-generation substrates evaluated for lactic acid production. 2G Substrate Strain °C Titer g/L Productivity g/L/h Yield g/g Isomer Reference Bagasse sulfite pulp B. coagulans CC-17 50 110.0 0.55 0.72 L Zhou et al., 2016 Brewers’ spent grains L. rhamnosus ATCC 7469 37 48.0 0.96 0.87 L Radosavljević et al., 2018 Coffee pulp B. coagulans 52 4.02 0.78 L Pleissner et al., 2016 Corn stover L. pentosus FL0421 37 92.3 1.92 0.66 Hu et al., 2016 B. coagulans NBRC 12714 50 92.0 13.8 0.91 L Ma et al., 2016 Corncob residue B. coagulans H-1 50 68.0 0.85 L Jiang et al., 2019 B. coagulans LA204 50 123.0 1.37 0.77 Zhang et al., 2016b B. coagulans IPE22 52 53.5 2.97 0.92 L Wang et al., 2018 Dried distiller’s grains L. coryniformis torquens DSM 20004 37 38.1 0.80 0.35 D Zaini et al., 2019 Food waste L. casei Shirota 37 94.0 2.61 0.94 Kwan et al., 2016 Streptococcus sp. A620 35 58.0 2.16 0.81 L Pleissner et al., 2017 Jackfruit seed powder Streptococcus sp. A620 37 109.0 L Nair et al., 2016 Oil palm empty fruit bunch B. coagulans JI12 50 120.0 4.30 0.49 L Juturu and Wu, 2018 Orange peel L. delbrueckii delbrueckii CECT 286 37 6.72 0.88 D de la Torre et al., 2019 L. casei 2246 37 209.6 g/kg 0.88 Ricci et al., 2019 Paper sludge L. rhamnosus ATCC 7469 37 108.2 0.62 Marques et al., 2017 S. flavescens residues and food waste L. casei CICC 6106 37 48.4 0.73 0.90 Zheng et al., 2017 Sugarcane bagasse L. plantarum CCT 3751 37 34.5 0.58 0.34 Oliveira et al., 2018c B. coagulans DSM 14-300 52 56.0 1.70 0.87 L Oliveira et al., 2019b B. coagulans DSM2314 50 91.7 0.92 0.94 L van der Pol et al., 2016 L. pentosus 37 72.7 1.01 0.61 Unrean 2018 Wheat straw B. coagulans MA-13 55 1.11 1.23 Aulitto et al., 2019 3. Genetic engineering microorganisms for 2G-lactic acid production Metabolic engineering of LAB presents a novel approach for re-routing metabolic reactions to produce desired compounds in higher amounts, such as LA, flavor compounds, sweeteners, exopolysaccharide, vitamins, among others (Sauer et al., 2017; Stefanovic et al., 2017). Interesting options for improving the phenotype and genotype of LAB are mutation, directed evolution, and genetic engineering (Cubas-Cano et al., 2018). The 225 rapid developments in genomics and its associated technologies have transformed the understanding of the diversity and functionality of LAB (McAuliffe, 2017). The interest in LAB genomes, considering them as important microbial cell factories started in the early ’90s (Stefanovic et al., 2017). However, their potential as microbial cell factories for the chemical industry is only emerging (Sauer et al., 2017). Besides of technology development, one of the reasons for this late development are concerns regarding the safety od genetically engineering microorganism for the environment and for the workers, as well as the acceptance of the general public to products from genetically engineered microorganisms. Regarding 2G-LA production, genetic engineering has concentrated on improving the LA fermentation parameters, enhancing the acid tolerance of production organisms and their abilities to utilize a broad range of substrates, including fermentable 2G-sugars (Upadhyaya et al., 2014). Substrates may represent one of the major costs in the LA production chain (Upadhyaya et al., 2014), one of the reasons why genetic engineering is expected to enhance and be a tool enabling 2G-LA production both, from lignocellulosic biomass and biomass-derived waste streams. The efforts are currently focused on increasing microbial resistance toward inhibitory compounds derived from biomass pretreatment and avoid carbon catabolite repression (CCR) when using mixed sugars for fermentation (Upadhyaya et al., 2014), always associated with high titers and high isomeric purities. Front this scenario, Table 2 present some recent examples of LAB genetic modifications towards currently challenges in the LA production chain. Table 2: Genetically engineering lactic acid bacteria for second-generation lactic acid production. Challenge addressed Approach Outcome Organism Substrate Reference Direct conversion of xylan to LA xynR8 expression Direct SSF xylan to produce LA in one step L. brevis Xylan Hu et al., 2011 D-LA fermentation from a mixture of xylose and glucose Introduction of xylAB operon into ldhL1 gene-deficient D-LA fermentation from mixed sugars without CCR L. plantarum Glucose Xylose Arabinose Yoshida et al., 2011 L-LA fermentation from xylose Disruption of ptk gene, introduction of xylRAB genes and insertion of tkt gene L-LA production from xylose with a high optical purity L. lactis Xylose Shinkawa et al., 2011 D-LA production from cellulosic feedstocks in SSF Introduction of xylAB operon and tkt gene in a ldhL1-deficient Increased D-LA production and productivity with high purity L. plantarum Hardwood pulp Hama et al., 2015 High tolerance to inhibitors and L-LA optically pure production Interruption of ldhD gene Improved L-LA production from lignocellulosic biomass L. paracasei Non- detoxified wood and rice straw Kuo et al., 2015 Improve L-LA production from crude glycerol Disruption of pflB gene and expression of the ldhL1LP gene High conversion of crude glycerol to L- LA E. faecalis Glycerol Doi, 2015 D-LA production from renewable resources xylose-assimilating genes cloned into an L-lactate-deficient strain Increased D-LA production with high purity L. plantarum Corn stover Zhang et al., 2016a High titer L and D-LA production from corn stover feedstock ldh/ldhD genes separately disrupted High titer L and D- LA P. acidilactici Detoxified corn stover Yi et al., 2016 CCR: carbon catabolite repression; SSF: simultaneous saccharification and fermentation 4. Conclusions 2nd generation lactic acid production still has challenges to overcome before becoming a world reality. However, the current scenario shows good perspectives of 2G-lactic acid production reach industrial-scale production, especially considering the biorefinery concept. The use of waste and/or lignocellulose material remaining from already consolidated industrial processes, especially in food and chemical production, opens the possibility of expanding sustainable and renewable processes to produce 2G-LA. With the possibility of 226 replacing petrochemical derivates and not competing with food industry, 2G-LA produced from already available material has not only an interesting economic appeal, but also a high social impact due to the growing consciences that it is important to reduce the world petrochemical dependence and change the current production model to a more sustainable one. 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