Abstract
For the first time, Lactobacillus plantarum strains carrying heterologous genes encoding multifunctional glycoside hydrolases were constructed and used as additives for alfalfa silage. The chemical characteristics, nonstructural carbohydrate composition, and fermentation quality of alfalfa silage were examined. The supernatant of L. plantarum expressing CbXyn10C and Bgxg1 (LP11AG) showed activities on xylan, Avicel, and carboxymethylcellulose (CMC), while the supernatant of the wild-type L. plantarum showed no activity. When LP11AG was used as silage additive, the water-soluble carbohydrate content of alfalfa silage increased by 72%, 55%, and 155% compared with control when the silage was stored at 20 °C, 30 °C, and 40 °C, respectively. With LP11AG being used as an additive for the alfalfa silage stored at 20 °C, the hemicellulose, cellulose, and acid detergent ligninin (ADL) contents decreased by 17%, 6%, and 14% compared with the control (p < 0.05), respectively. Compared with the corresponding original contents, the contents of glucose, arabinose, galactose, and fructose detected in silage treated with LP11AG after 45 days of ensiling increased by 55%, 1494%, 68%, and 5% , respectively, when stored at 40 °C. Raffinose and stachyose, originally present in alfalfa, disappeared after ensiling. In conclusion, our results suggest that LP11AG provides a substantial benefit as a silage additive.
Similar content being viewed by others
References
Armentano LE, Pastore SC, Hoffman PC (1988) Particle size reduction of alfalfa silage did not alter nutritional quality of high forage diets for dairy cattle. J Dairy Sci 71(2):409–413. https://doi.org/10.3168/jds.S0022-0302(88)79570-3
Arshad M, Feyissa BA, Amyot L, Aung B, Hannoufa A (2017) MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by silencing SPL13. Plant Sci 258:122–136. https://doi.org/10.1016/j.plantsci.2017.01.018
Borrero J, Jimenez JJ, Gutiez L, Herranz C, Cintas LM, Hernandez PE (2011) Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria. J Biotechnol 156(1):76–86. https://doi.org/10.1016/j.jbiotec.2011.07.038
Brunecky R, Alahuhta M, Xu Q, Donohoe BS, Crowley MF, Kataeva IA, Yang SJ, Resch MG, Adams MW, Lunin VV, Himmel ME, Bomble YJ (2013) Revealing nature’s cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA. Science 342:1513–1516. https://doi.org/10.1126/science.1244273
Coblentz WK, Muck RE, Borchardt MA, Spencer SK, Jokela WE, Bertram MG, Coffey KP (2014) Effects of dairy slurry on silage fermentation characteristics and nutritive value of alfalfa. J Dairy Sci 97(11):7197–7211. https://doi.org/10.3168/jds.2014-8582
Dean DB, Adesogan AT, Krueger N, Littell RC (2005) Effect of fibrolytic enzymes on the fermentation characteristics, aerobic stability, and digestibility of bermudagrass silage. J Dairy Sci 88(3):994–1003. https://doi.org/10.3168/jds.s0022-0302(05)72767-3
Denek N, Can A, Avci M, Aksu T, Durmaz H (2011) The effect of molasses-based pre-fermented juice on the fermentation quality of first-cut lucerne silage. Grass Forage Sc 66 (2):243-250. https://10.1111/j.1365-2494.2011.00783.x
Desta ST, Yuan X, Li J, Shao T (2016) Ensiling characteristics, structural and nonstructural carbohydrate composition and enzymatic digestibility of Napier grass ensiled with additives. Bioresour Technol 221:447–454. https://doi.org/10.1016/j.biortech.2016.09.068
Ding W, Guo X, Ataku K (2014) Characterization of peptides in ensiled alfalfa treated with different chemical additives. Anim Sci J 84(12):774–781. https://doi.org/10.1111/asj.12065
Japan Grassland Farming Forage Seed Association (1994) Guide book for quality evaluation of forage, Tokyo, pp 82–87
Ke WC, Ding WR, Xu DM, Ding LM, Zhang P, Li FD, Guo XS (2017) Effects of addition of malic or citric acids on fermentation quality and chemical characteristics of alfalfa silage. J Dairy Sci 100(11):8958–8966. https://doi.org/10.3168/jds.2017-12875
Kim SK, Chung D, Himmel ME, Bomble YJ, Westpheling J (2017) Engineering the N-terminal end of CelA results in improved performance and growth of Caldicellulosiruptor bescii on crystalline cellulose. Biotechnol Bioeng 114(1):945–950. https://doi.org/10.1002/bit.26242
Li J, Yuan X, Desta ST, Dong Z, Mugabe W, Shao T (2018a) Characterization of Enterococcus faecalis JF85 and Enterococcus faecium Y83 isolated from Tibetan yak (Bos grunniens) for ensiling Pennisetum sinese. Bioresour Technol 257:76–83. https://doi.org/10.1016/j.biortech.2018.02.070
Li J, Yuan X, Dong Z, Mugabe W, Shao T (2018b) The effects of fibrolytic enzymes, cellulolytic fungi and bacteria on the fermentation characteristics, structural carbohydrates degradation, and enzymatic conversion yields of Pennisetum sinese silage. Bioresour Technol 264:123–130. https://doi.org/10.1016/j.biortech.2018.05.059
Liu JR, Yu B, Zhao X, Cheng KJ (2007) Coexpression of rumen microbial beta-glucanase and xylanase genes in Lactobacillus reuteri. Appl Microbiol Biotechnol 77(1):117–124. https://doi.org/10.1007/s00253-007-1123-5
Ma X, Wang G, Li D, Hao Y (2016) Microcin v production in Lactobacillus plantarum LB-B1 using heterologous leader peptide from pediocin pa-1. Curr Microbiol 72(3):357–362. https://doi.org/10.1007/s00284-015-0927-2
Mathiesen G, Sveen A, Brurberg MB, Fredriksen L, Axelsson L, Eijsink VG (2009) Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1. BMC Genomics 10(1):425. https://doi.org/10.1186/1471-2164-10-425
Mierau I, Kleerebezem M (2005) 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68(6):705–717. https://doi.org/10.1007/s00253-005-0107-6
Morais S, Shterzer N, Lamed R, Bayer EA, Mizrahi I (2014) A combined cell-consortium approach for lignocellulose degradation by specialized Lactobacillus plantarum cells. Biotechnol Biofuels 7(1):112. https://doi.org/10.1186/1754-6834-7-112
Morrison JM, Elshahed MS, Youssef N (2016) A multifunctional GH39 glycoside hydrolase from the anaerobic gut fungus Orpinomyces sp. strain C1A. Peer J 4(3):e2289. https://doi.org/10.7717/peerj.2289
Muck RE, Nadeau EMG, McAllister TA, Contreras-Govea FE, Santos MC, Kung L Jr (2018) Silage review: recent advances and future uses of silage additives. J Dairy Sci 101(5):3980-4000. https://doi.org/10.3168/jds.2017-13839
Nadeau EM, Buxton DR, Russell JR, Allison MJ, Young JW (2000) Enzyme, bacterial inoculant, and formic acid effects on silage composition of orchardgrass and alfalfa. J Dairy Sci 83(7):1487–1502. https://doi.org/10.3168/jds.S0022-0302(00)75021-1
Nguyen STC, Freund HL, Kasanjian J, Berlemont R (2018) Function, distribution, and annotation of characterized cellulases, xylanases, and chitinases from CAZy. Appl Microbiol Biotechnol 102(4):1629–1637. https://doi.org/10.1007/s00253-018-8778-y
Ni K, Zhao J, Zhu B, Su R, Pan Y, Ma J, Zhou G, Tao Y, Liu X, Zhong J (2018) Assessing the fermentation quality and microbial community of the mixed silage of forage soybean with crop corn or sorghum. Bioresour Technol 265:563–567. https://doi.org/10.1016/j.biortech.2018.05.097
Nsereko VL, Rooke JA (2000) Characterisation of peptides in silages made from perennial ryegrass with different silage additives. J Sci Food Agric 80(6):725-731. https://doi.org/10.1002/(SICI)1097-0010(20000501)80:6<725::AID-JSFA602>3.0.CO;2-8
Ozkose E, Akyol I, Kar B, Comlekcioglu U, Ekinci MS (2009) Expression of fungal cellulase gene in Lactococcus lactis to construct novel recombinant silage inoculants. Folia Microbiol 54(4):335–342. https://doi.org/10.1007/s12223-009-0043-4
Peng X, Su H, Mi S, Han Y (2016) A multifunctional thermophilic glycoside hydrolase from Caldicellulosiruptor owensensis with potential applications in production of biofuels and biochemicals. Biotechnol Biofuels 9(1):98. https://doi.org/10.1186/s13068-016-0509-y
Rossi F, Rudella A, Marzotto M, Dellaglio F (2001) Vector-free cloning of a bacterial endo-1,4-β-glucanase in lactobacillus plantarum and its effect on the acidifying activity in silage: use of recombinant cellulolytic lactobacillus plantarum as silage inoculant. Antonie Van Leeuwenhoek 80(2):139-147. https://doi.org/10.1023/A:1012223220427
Rud I, Jensen PR, Naterstad K, Axelsson L (2006) A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiology 152(Pt 4):1011-1019. https://doi.org/10.1099/mic.0.28599-0
Shao Q, Chundawat SP, Krishnan C, Bals B, Sousa Lda C, Thelen KD, Dale BE, Balan V (2010) Enzymatic digestibility and ethanol fermentability of AFEX-treated starch-rich lignocellulosics such as corn silage and whole corn plant. Biotechnol Biofuels 3(1):12. https://doi.org/10.1186/1754-6834-3-12
Sheperd AC, Maslanka M, Quinn D, Kung L Jr (1995) Additives containing bacteria and enzymes for alfalfa silage. J Dairy Sci 78(3):565–572. https://doi.org/10.3168/jds.S0022-0302(95)76667-X
Smith LH (1962) Theoretical carbohydrates requirement for alfalfa silage production1. Agron J 54 (4):291. https://10.2134/agronj1962.00021962005400040003x
Talamantes D, Biabini N, Dang H, Abdoun K, Berlemont R (2016) Natural diversity of cellulases, xylanases, and chitinases in bacteria. Biotechnol Biofuels 9:133. https://doi.org/10.1186/s13068-016-0538-6
Tengerdy RP, Weinberg ZG, Szakacs G, Wu M, Linden JC, Henk LL, Johnson DE (2010) Ensiling alfalfa with additives of lactic acid bacteria and enzymes. J Sci Food Agric 55(2):215-228. https://doi.org/10.1002/jsfa.2740550207
Tian J, Li Z, Yu Z, Zhang Q, Li X (2017) Interactive effect of inoculant and dried jujube powder on the fermentation quality and nitrogen fraction of alfalfa silage. Anim Sci J 88(4):633-642. https://doi.org/10.1111/asj.12689
Tisma M, Planinic M, Bucic-Kojic A, Panjicko M, Zupancic GD, Zelic B (2018) Corn silage fungal-based solid-state pretreatment for enhanced biogas production in anaerobic co-digestion with cow manure. Bioresour Technol 253:220–226. https://doi.org/10.1016/j.biortech.2018.01.037
Xue X, Wang R, Tu T, Shi P, Ma R, Luo H, Yao B, Su X (2015) The N-terminal gh10 domain of a multimodular protein from Caldicellulosiruptor bescii is a versatile xylanase/β-glucanase that can degrade crystalline cellulose. Appl Environ Microbiol 81:3823–3833. https://doi.org/10.1128/AEM.00432-15
Yuan XJ, Wen AY, Wang J, Desta ST, Dong ZH, Shao T (2018) Effects of four short-chain fatty acids or salts on fermentation characteristics and aerobic stability of alfalfa (Medicago sativa L.) silage. J Sci Food Agric 98(1):328–335. https://doi.org/10.1002/jsfa.8475
Zhang Q, Yu Z (2017) Characterization, identification and application of lactic acid bacteria isolated from Leymus chinensis silage. Grassl Sci 63(2):111–117. https://doi.org/10.1111/grs.12156
Zhang Q, Yu Z, Wang X, Tian J (2018) Effects of inoculants and environmental temperature on fermentation quality and bacterial diversity of alfalfa silage. Anim Sci J 89(8):1085–1092. https://doi.org/10.1111/asj.12961
Funding
This work was supported by the Modern Agro-industry Technology Research System (CARS-34), the National Natural Science Foundation of China (31702181), the National Key Research and Development Program of China (2017YFD0502100), and Fundamental Research Funds for the Central Universities (2018QC048).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
This article does not contain any studies with human participants or animal experiments.
Conflict of interest
The authors declare that they have no conflicts of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 979 kb)
Rights and permissions
About this article
Cite this article
Guo, J., Xie, Y., Yu, Z. et al. Effect of Lactobacillus plantarum expressing multifunctional glycoside hydrolases on the characteristics of alfalfa silage. Appl Microbiol Biotechnol 103, 7983–7995 (2019). https://doi.org/10.1007/s00253-019-10097-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-019-10097-6