Biochemical conversion of sweet sorghum bagasse to succinic acid Enlin Lo, 1 Luiza Brabo-Catala, 2 Ioannis Dogaris, 2 Ehab M. Ammar, 2, 3 and George P. Philippidis 2, * Department of Chemical and Biomedical Engineering, University of South Florida, 4202 E. Fowler Avenue, Tampa, FL 33620, USA, 1 Patel College of Global Sustainability, University of South Florida, 4202 E. Fowler Avenue, Tampa, FL 33620, USA, 2 and Genetic Engineering and Biotechnology Research Institute, University of Sadat City, El-Sadat City, Egypt 3 Received 23 May 2019; accepted 20 July 2019 Available online xxx Succinic acid, an important intermediate in the manufacture of plastics and other commodity and specialty chemicals, is currently made primarily from petroleum. We attempted to biosynthesize succinic acid through microbial fermen- tation of cellulosic sugars derived from the bagasse of sweet sorghum, a renewable feedstock that can grow in a wide range of climates around the world. We investigated pretreating sweet sorghum bagasse (SSB) with concentrated phosphoric acid at mild conditions (40e85  C) at various residence times and biomass concentrations. We then subjected the pretreated SSB to enzymatic hydrolysis with a commercial cellulase to release glucose. The highest glucose yield was obtained when SSB was pretreated at 50  C for 43 min at 130 g/L biomass concentration on dry basis. Fermentation was carried out with Actinobacillus succinogenes 130Z, which readily converted 29.2 g/L of cellulosic glucose to 17.8 g/L of succinic acid in a 3.5-liter bioreactor sparged with CO 2 at a rate of 0.5 vvm, thus reducing the carbon footprint of the process. Overall, we demonstrated, for the first time, the use of SSB for production of succinic acid using practices that lower energy use, future equipment cost, waste generation, and carbon footprint. Ó 2019, The Society for Biotechnology, Japan. All rights reserved. [Key words: Succinic acid; Sweet sorghum bagasse; Acid pretreatment; Phosphoric acid; Biochemical conversion] Succinic acid, a four-carbon dicarboxylic acid used in a wide range of applications in the chemical, food, and pharmaceutical industries (1), is currently produced almost exclusively from oil. Sustainability concerns about the use of fossil sources and the en- ergy intensity of high-temperature and high-pressure manufacturing processes have created interest in producing suc- cinic acid via fermentation of sugars (2). However, use of sugars derived from food crops, like corn and sugarcane, raises significant societal concerns about sustainability, especially regarding food security and land use change. To enhance the sustainability of fermentative succinic acid production, we investigated the use of sweet sorghum bagasse (SSB) as a renewable resource in a biochemical process that in- corporates sustainability practices. Sweet sorghum is a promising source of sugar and fiber thanks to its ability to grow in a wider range of climates and soils and survive drought conditions compared to sugarcane, while its cellulosic fiber can be converted to ethanol (3,4). SSB is currently employed as animal feed, soil fertilizer, and solid fuel for power generation (5). However, it can be further valorized through chemical pretreatment, making it a po- tential source of cellulosic sugars and eventually of valuable bio- products via enzymatic hydrolysis and fermentation (6,7). Although several lignocellulosic species, such as pinewood, corn stover, and sugarcane bagasse, have been used before for fermen- tative succinic acid production (8,9), SSB has not been investigated, prompting us to explore its potential as a renewable feedstock for succinic acid biosynthesis. Importantly, previous work on succinic acid production from sweet sorghum utilized the juice as feedstock, but no attempts have been reported on using its bagasse (10,11). Chemical processing of biomass at large scale is mostly practiced with strong acids, primarily sulfuric. Although effective in condi- tioning biomass, such processes require high temperatures (160e240  C) and pressures (w10 atm) (12e14), thus necessitating high energy consumption and use of expensive materials of con- struction for the required equipment. Furthermore, the use of sul- furic acid results in the generation of significant amounts of gypsum, an environmental liability, during acid neutralization following pretreatment (14). Steering towards green chemistry practices, we examined SSB pretreatment under much milder conditions with concentrated phosphoric acid (15,16), a weak acid that is much less hazardous and corrosive compared to sulfuric acid (17). Importantly, neutralization of phosphoric acid at the end of the pretreatment process results in the formation of phosphate salts, which can serve as a fertilizer co-product, as opposed to gypsum waste generated by sulfuric acid. Moreover, the phosphates can serve as buffer and nutrient during microbial fermentation processes (18). Chemically pretreated biomass is amenable to enzymatic hy- drolysis using cellulolytic enzymes, which depolymerize cellulose to fermentable glucose (19e21). A number of bacterial and yeast strains are known to ferment cellulosic glucose to succinic acid and other chemicals and biofuels (22e24). We elected to use A. succinogenes 130Z, a promising succinic acid producer (25) with an interesting sustainability feature: whereas it anaerobically produces a mixture of organic acids and ethanol (25,26), when exposed to a CO 2 -enriched environment its metabolism favors succinic acid formation, as glucose-derived glyceraldehyde (3- carbon molecule) is converted to succinic acid (4-carbon * Corresponding author. Tel.: þ1 813 974 9333; fax: þ1 813 974 2522. E-mail address: gphilippidis@usf.edu (G.P. Philippidis). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. xxx No. xxx, xxx, xxxx 1389-1723/$ e see front matter Ó 2019, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2019.07.003 Please cite this article as: Lo, E et al., Biochemical conversion of sweet sorghum bagasse to succinic acid, J. Biosci. Bioeng., https://doi.org/ 10.1016/j.jbiosc.2019.07.003