Efficient Simultaneous Saccharification and Fermentation of Inulin

21 2,3-Butanediol (2,3-BD) is an important starting material for the manufacture of bulk 22 chemicals. For efficient and large-scale production of 2,3-BD through fermentation, 23 low-cost substrates are required. One such substrate, inulin, is a polydisperse fructan 24 found in a wide variety of plants. In this study, a levanase with high inulinase activity 25 and high pH and temperature stability was identified in a Bacillus licheniformis strain 26 ATCC14580. The B. licheniformis strain ATCC14580 was found to efficiently 27 produce 2,3-BD from fructose at 50°C. Then, the levanase was used for simultaneous 28 saccharification and fermentation (SSF) of inulin to 2,3-BD. A fed-batch SSF yielded 29 103.0 g/L 2,3-BD in 30 h, with a high productivity of 3.4 g/L·h. The results suggest 30 that the SSF process developed with the used thermophilic B. licheniformis strain 31 might be a promising alternative for efficient 2,3-BD production from the favorable 32 substrate inulin. 33 34 on D ecem er 8, 2017 by gest ht://aem .sm .rg/ D ow nladed fom 2,3-Butanediol (2,3-BD) is used as a starting material for the manufacture of bulk 35 chemicals such as methyl ethyl ketone and 1,3-butadiene (1, 2). With a heating value 36 of 27,200 J/g, 2,3-BD compares favorably with ethanol (29,100 J/g) and methanol 37 (22,100 J/g) as a liquid fuel or fuel additive (3). 2,3-BD can be produced by chemical 38 or biotechnological methods (1, 2). Due to the gradual exhaustion of crude oil 39 reserves, interest in the biotechnological production of 2,3-BD has increased greatly 40 in recent years. Currently, Klebsiella pneumoniae, K. oxytoca, Serratia marcescens, 41 and Bacillus licheniformis are regarded as the most efficient 2,3-BD producers (4-7). 42 However, in these studies refined glucose and sucrose were used as substrates, which 43 may not be economically feasible. Because the cost of raw materials accounts for 44 more than 30% of the total production costs of fermentation, low-price substrates and 45 efficient strains are required for 2,3-BD production (8). Therefore, from an economic 46 point of view, cheap raw materials, such as lignocellulosic materials, corncob 47 molasses, corncob acid hydrolysates, starch hydrolysate, whey permeate, sugarcane 48 molasses, and cassava powder, could be used for 2,3-BD production. However, the 49 productivity and concentration of 2,3-BD obtained by using these cheap substrates are 50 still low (1). Thus, it is desirable to identify cheap, non-food, renewable sources, and 51 develop appropriate fermentation strategies to obtain high 2,3-BD productivity and 52 concentrations. 53 Fructans are the most abundant nonstructural polysaccharides found in plants after 54 starch, and they are found in wide variety of plants. One of these fructans, inulin, is a 55 polydisperse fructan consisting mainly of β-(2,1)-D-fructosyl-fructose links 56 on D ecem er 8, 2017 by gest ht://aem .sm .rg/ D ow nladed fom terminated by a sucrose residue (9). It serves as a storage polysaccharide in many 57 plants of the Compositae and Gramineae, and accumulates in the underground roots 58 and tubers of several plants, including Vernonia herbacea, Jerusalem artichoke 59 (Helianthus tuberosus), chicory, and dahlia (10). Of these plants, Jerusalem artichoke 60 is interesting because of its good productivity (50 tons per hectare) and its resistance 61 to low temperatures and disease. It can even be cultivated in sandy soils and tideland, 62 requiring limited human intervention for cultivation, and thus does not compete for 63 arable lands with grain crops (1). Inulin constitutes up to 85% of the dry weight of the 64 Jerusalem artichoke (11) and 16% of the wet basis of chicory root (12). The 65 worldwide production of inulin is currently estimated to be about 350,000 tons (13). 66 Although its total quantity is not comparable to that of lignocellulose biomass, inulin 67 could be easily processed by using currently available technologies, and it may even 68 be easier to process than starch (11). Thus, inulin has recently received much attention 69 as a renewable feedstock for the production of fructose and chemicals, such as ethanol 70 and lactic acid (14–16). However, there have been few studies on 2,3-BD production 71 from inulin as substrate (17, 18). 72 Inulin can be hydrolyzed to fructose and glucose by inulinase or levanase. Both 73 inulinase and levanase are fructofuranosyl hydrolases that are produced by a wide 74 variety of organisms, including plants, bacteria, molds, and yeasts (13). Although 75 many inulinases and levanases have been reported, the commercial inulinase, an 76 inulinase from Aspergillus niger, is expensive (Sigma Product ID 16285, Novozym 77 960; $188 for 250 mL), and there is no commercial levanase. Dao et al. suggested that 78 on D ecem er 8, 2017 by gest ht://aem .sm .rg/ D ow nladed fom a commercial glucoamylase (GA-L New from Genencor) could be used for the 79 hydrolysis of Jerusalem artichoke tubers. However, the optimum pH of glucoamylase 80 for inulin hydrolysis is 4.0, and the stability of this enzyme decreased significantly 81 when the pH was increased to 5.6 (19). These characteristics make GA-L New 82 unsuitable for simultaneous saccharification and fermentation (SSF) of 2,3-BD, 83 because the pH range for optimal 2,3-BD production is generally between 5.5–7.0 (3). 84 Therefore, identification of an appropriate inulinase or levanase is a key step for 85 industrial-scale production of 2,3-BD from inulin. 86 Most of the reported inulinases and levanases have optimum temperatures in the 87 range of 45°C–55°C (13). However, many microbes, including K. pneumoniae, K. 88 oxytoca, S. marcescens, and Paenibacillus polymyxa, have the ability to produce 89 2,3-BD at 28°C–37°C (1). Utilization of these mesophilic strains for SSF of inulin to 90 2,3-BD may increase the dosage of inulinase or levanase; therefore, the SSF process 91 with a thermophilic 2,3-BD-producer may use lower dosage of the enzyme. In our 92 previous study, we used a thermophilic bacterium B. licheniformis 10-1-A for 2,3-BD 93 production at an optimum temperature of 50°C (7); thus thermophilic B. licheniformis 94 might be suitable for SSF of inulin to 2,3-BD under the 50°C fermentative conditions. 95 In this study, an another thermophilic B. licheniformis strain ATCC14580 was used to 96 produce 2,3-BD from inulin. In addition, a gene in B. licheniformis ATCC14580 97 encoding a levanase was expressed, purified, and partially characterized. Efficiencies 98 of various fermentation strategies, including SSF and separate hydrolysis and 99 fermentation (SHF), were investigated. 100 on D ecem er 8, 2017 by gest ht://aem .sm .rg/ D ow nladed fom