Paenibacillus panacisoli enhances growth of Lactobacillus spp. by producing xylooligosaccharides in corn stover ensilages
Graphical abstract
Introduction
Ensiling is a crop preservation method based on natural lactic acid fermentation under anaerobic conditions (Gollop, Zakin, & Weinberg, 2005). It is also a complex process that involves the interaction of numerous microbial species, ultimately changing the biochemical composition of silages (McDonald, Henderson, & Heron, 1991). Among the microbial species, lactic acid bacteria (LAB) are the most important group of bacteria which convert the water soluble carbohydrates (WSC) into lactic acid and acetic acid to inhibit the undesirable microorganisms (Rooke, & Hatfield, 2003). Thus, homofermentative as well as heterofermentative LAB species with excellent performance, such as Lactobacillus plantarum and Lactobacillus brevis, have been used as bacterial inoculates for silages (Francisco, Richard, Glen, Glen, & Paul, 2013; Reich, & Kung, 2000). However, LAB are lacking in lignocellulosic biomass-degrading enzymes which degrade lignocellulosic component into WSC (Sun et al., 2012).
The ensiling of corn stover is gaining increasing attention due to its high nutritional value and good palatability (Pang et al., 2011). Corn stover (particular wilted corn stover) is mainly composed of cellulose, hemicellulose and lignin, but a small amount of WSC (Ribeiro et al., 2015), which limits the growth of LAB during ensilage. Hemicellulose is the second abundant organic materials and takes up 25.1% dry matter of corn stover (Lizasoain et al., 2017), and xylan is the most important hemicellulose component (Liao et al., 2014). Xylanases are key enzymes for degradation of the backbone of xylan by hydrolyzing β-1, 4-xylosidic linkages between two d-xylopranosyl residues, and release of WSC (Shallom, & Shoham, 2003; Yan, Hao, Jiang, Zhai, & Chen, 2009). Hence, xylanases are often used as additives in ensilages. The hydrolysis abilities of xylanases on forage crops such as corn and alfalfa in ensilages were determined previously and they were considered as the promising candidates for increasing in vitro NDF digestibility (Lynch, Prema, Van Hamme, Church, & Beauchemin, 2014; Yang, Chen, Gao, & Li, 2001). In consequence, the fiber content of ensiled forage crops is reduced, so as to lighten the digestion burden of animals. Meanwhile, the produced WSC can promote the growth of LAB. So far, the commercial xylanases used in silage processes are from fungi, a group of aerobic microorganisms that could not grow in silage processes (Khota, Pholsen, Higgs, & Cai, 2016). The fermentation and extraction process of xylanase preparation inevitably increase costs.
Paenibacillus spp., widely distributed in soil and associated with plants roots, are important strains in sustainable agriculture biotechnology (Grady, MacDonald, Liu, Richman, & Yuan, 2016; Kämpfer et al., 2017). Owing to promoting the growth of plants and protecting plants against insect herbivores and phytopathogens, multiple species of Paenibacillus have been used in commercial biofertilizers (Weselowski, Nathoo, Eastman, MacDonald, & Yuan, 2016). Moreover, specific Paenibacillus strains also produce a variety of enzymes, including amylase, cellulases, xylanases and lipases, with potential applications in process manufacturing for food, textile, paper and biofuel (Raddadi, Cherif, Daffonchio, & Fava, 2013; Valenzuela, Diaz, & Pastor, 2014). Meanwhile, Paenibacillus strains and spores are found involving in ensilages, and the species can survive in the late phase of ensiling fermentation (Driehuis, Hoolwerf, & Rademaker, 2016; Ning et al., 2017). However, no research into the performance of these species and their enzymes in ensiling process has been conducted yet.
P. panacisoli is a newly identified facultative anaerobic bacteria (Ten et al., 2006). In this study, a strain P. panacisoli SDMCC050309 with high xylanase activities was isolated from ensiled corn stover. We characterized the xylanases properties produced by P. panacisoli SDMCC050309, and investigated the effects of this strain and its hydrolysates on L. brevis SDMCC050297 and L. parafarraginis SDMCC050300. The aim of this study is to find out the effects of xylanase-producing microbes on Lactobacillus inoculant strains in corn stover ensilages.
Section snippets
Strains, vectors, culture conditions and chemicals
To isolate bacteria with xylanase activity, LB (Luria-Bertani) medium supplementary with 0.5% larch wood xylan was used. The isolates were cultured in LB medium at 37 °C aerobically. The modified minimal medium: (g/L) K2HPO4, 1.67; KH2PO4, 0.87; NaCl, 0.05; MgSO4·7H2O, 0.1; CaCl2, 0.04; FeCl3, 0.004; Na2MoO4·2H2O, 0.005; biotin, 0.01; nicotinic acid, 0.02; pantotenic acid, 0.01; NH4Cl, 1, and supplemented with 0.1% yeast extract and 0.5% larch wood xylan (MM-LX medium) or 0.5% corn stover bran
Isolation and identification of xylan-degrading bacteria
Among all the isolates, one strain SDMCC050309 showed maximal hydrolysis zone in the plates containing larch wood xylan, suggesting it was a xylanase producer (data not shown). To identify the strain, 16S rDNA gene was amplified and sequenced. A neighbor-joining phylogenetic tree based on this sequence was constructed as shown in Fig. 1. Based on the morphological and physiological characteristics, combined with the 16S rRNA gene sequence (shared 99% sequence identity with P. panacisoli DSM
Discussion
LAB and lignocellulosic biomass-degrading microbes are naturally adhered to the plant surface (Kadivar & Stapleton, 2003). During the ensiling process, they co-exist in forage crops to carry out the fermentation. Although LAB dominate the fermentation in well-processed silage, producing the low pH conditions that to stop the growth and metabolism of other microorganisms, there are diverse microbial activities in the initial period of ensiling (Dunièrea, Sindoub, Chaucheyras-Durand, Chevallier,
Conclusion
During the ensiling process, LAB and other microorganisms co-exist in forage crops to carry out the fermentation. However, the knowledge about the association of lignocellulosic biomass-degrading microbes with the LAB is still limited. Two xylanases produced by P. panacisoli SDMCC050309 were identified and characterized. Their hydrolysis products XOS could act as carbon source for promoting the growth of L. brevis SDMCC050297 and L. parafarraginis SDMCC050300 to generate high level of acetic
Acknowledgements
This study was funded by Public Service Sectors (Agriculture) Special and Scientific Research Projects (201503134), National Natural Science Foundation of China (31471715), and National Science Foundation for Young Scientists of China (31400077).
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