3.5 Succinic acid production from corn cob using co-cultivation system
Corn cob was considered to be rich in hemicellulose (Ma et al., 2016), hence, direct conversion of corn cob through CBP was investigated for succinic acid production. In this study, corn cob containing approximately 33% of hemicellulose was directly used as substrate for succinic acid fermentation with only mechanic crush. As shown in Fig. 6A, the highest succinic acid concentration of 12.51 g/L was obtained from 80 g/L of corn cob after 8 days, representing the yield of 0.16 g/g. Before the dosage of strain 130Z, xylose continued to be accumulated and finally reached up to 8.68 g/L. In addition, xylose concentration was maintained at 7-8 g/L during co-cultivation process, while xylanase and β-xylosidase activities were maintained at 0.37-0.42 and 5.98-7.21 U/mL, respectively (Fig 6B).
Results showed that strain M5 and 130Z could be good partners to achieve succinic acid directly from hemicellulosic materials through CBP. The stable co-existence of these two strains was mainly based on competitive cheater and cooperator interactions (Shahab et al., 2018). When cooperative individuals benefit more from cooperative behavior than on-cooperators (cheaters), they can generally maintain cooperative phenotypes, referred to as assortment (Hol et al., 2013). In this study, strain M5 continuously secreted xylanase and β-xylosidase, which was a cooperative feature because xylose obtained by enzymatic hydrolysis was available as public goods for both strains. Strain 130Z may act as a cheater, because strain 130Z only utilized xylose for succinic acid production without contributing energy to the secretion of enzymes. However, strain M5 also benefited from the presence of strain 130Z, mainly reflected by the continuous consumption of xylose by strain 130Z, which could relieve product feedback inhibition to xylanase and β-xylosidase caused by high concentration of xylose (Hol et al., 2013).
3.6 Comparison of succinic production fromlignocellulosic biomass with other studies
So far, researchers have done series of work to realize the conversion of lignocellulosic biomass to succinic acid (Table 3). However, majority work involved complex pretreatment processes, such as biological, chemical, physical or physicochemical methods etc. For example, corn fiber could be pretreated with aqueous ammonia (SAA), dilute sulfuric acid and lignocellulosic enzymes into fermentable monomeric sugars for succinic acid production (Yoo, Nghiem, & Kim, 2016). 26.3 g/L of succinic acid with yield of 0.53 g/g could be produced from corn fiber hydrolysate by Escherichia coli AFP184. In addition, 30.00 g/L succinic acid with yield of 0.69 g/g was also obtained from dilute-acid pretreated corn stover hydrolysate by using Basfia succiniciproducens (Salvachua et al., 2016). Similarly, a maximum succinic acid titer and yield of 39.60 g/L and 0.78 g/g were achieved from deacetylated and dilute-acid pretreated corn stover hydrolysate byA. succinogenes (Bradfield et al., 2015). Recently, the enzymatic extracellular mixtures of two new isolates were shown to hydrolyze both cellulose and xylan into monosaccharides. The produced monosaccharides were converted into succinic acid ranging from 15 to 18 g/L by B. succiniciproducens BPP7, with an average yield 0.75 g/g (Pennacchio et al., 2018). All above examples were accompanied by complex and expensive pretreatment steps. Particularly, efforts have also been made to use genetic engineering to transform single bacterium with capabilities of lignocellulose degradation and succinic acid production. For example, an E. coli was engineered to secrete hemicellulases for succinate acid production from beechwood xylan through CBP (Zheng et al., 2012). 14.44 g/L of succinic acid with yield of 0.37 g/g was finally produced from 1% xylose and 3% xylan. This represents the first example CBP strain for succinic acid production from xylan without using externally supplied hydrolases. In this study, the designed synthetic microbial co-cultivation system can achieve higher succinic acid production without the need for complex genetic engineering and pretreatment process. The application of co-cultivation-based CBP may pave the way for direct lignocellulosic biomass conversion into biochemicals and bioenergy.
  1. CONCLUSIONS
In this study, a synthetic microbial co-cultivation system was designed to convert lignocellulosic biomass to succinic acid through CBP. Response surface methodology (RSM) was used to improve succinic acid titer. After process optimization, T. thermosaccharolyticum M5 and A. succinogenes 130Z produced up to 32.50 g/L of succinic acid with yield of 0.41 g/g from 80 g/L xylan w. In addition, 12.51 g/L succinic acid was also obtained from 80 g/L of corn cob, representing the first successful example for succinic acid production from lignocellulose by using microbial consortium. Future work was still needed to improve the hydrolytic rate to match the reducing sugar releasing and take up rates.