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      A Novel Triculture System (CC3) for Simultaneous Enzyme Production and Hydrolysis of Common Grasses through Submerged Fermentation

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          Abstract

          The perennial grasses are considered as a rich source of lignocellulosic biomass, making it a second generation alternative energy source and can diminish the use of fossil fuels. In this work, four perennial grasses Saccharum arundinaceum, Panicum antidotale, Thysanolaena latifolia, and Neyraudia reynaudiana were selected to verify their potential as a substrate to produce hydrolytic enzymes and to evaluate them as second generation energy biomass. Here, cellulase and hemi-cellulase producing three endophytic bacteria ( Burkholderia cepacia BPS-GB3, Alcaligenes faecalis BPS-GB5 and Enterobacter hormaechei BPS-GB8) recovered from N. reynaudiana and S. arundinaceum were selected to develop a triculture (CC3) consortium. During 12 days of submerged cultivation, a 55–70% loss in dry weight was observed and the maximum activity of β-glucosidase (5.36–12.34 IU) and Xylanase (4.33 to 10.91 IU) were observed on 2nd and 6th day respectively, whereas FPase (0.26 to 0.53 IU) and CMCase (2.31 to 4.65 IU) showed maximum activity on 4th day. Around 15–30% more enzyme activity was produced in CC3 as compared to monoculture (CC1) and coculture (CC2) treatments, suggested synergetic interaction among the selected three bacterial strains. Further, the biomass was assessed using Fourier-transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). The FTIR analysis provides important insights into the reduction of cellulose and hemicellulose moieties in CC3 treated biomass and SEM studies shed light into the disruption of surface structure leading to access of cellulose or hemicelluloses microtubules. The hydrolytic potential of the CC3 system was further enhanced due to reduction in lignin as evidenced by 1–4% lignin reduction in biomass compositional analysis. Additionally, laccase gene was detected from A. faecalis and E. hormaechei which further shows the laccase production potential of the isolates. To our knowledge, first time we develop an effective endophytic endogenous bacterial triculture system having potential for the production of extracellular enzymes utilizing S. arundinaceum and N. reynaudiana as lignocellulosic feedstock.

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          Bacterial endophytes in agricultural crops

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            Interlaboratory testing of methods for assay of xylanase activity

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              Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN.

              Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                31 March 2016
                2016
                : 7
                Affiliations
                1Molecular Microbiology and Systematics Laboratory, Department of Biotechnology, Mizoram University Aizawl, India
                2Department of Biotechnology, J.J College for Arts and Science Pudukkottai, India
                3Biocatalysts Lab, Department of Agricultural Microbiology, Tamil Nadu Agricultural University Coimbatore, India
                4Molecular Glyco-biotechnology Group, Department of Biochemistry, National University of Ireland Galway Galway, Ireland
                5Biotechnology Division, CSIR-North East Institute of Science and Technology Jorhat, Assam, India
                6Biochemical Engineering Research and Process Development Centre (BERPDC), Institute of Microbial Technology Chandigarh, India.
                Author notes

                Edited by: Weiwen Zhang, Tianjin University, China

                Reviewed by: Fu-Li Li, Chinese Academy of Sciences, China; Rajeeva Gaur, Dr. Ram Manohar Lohia Avadh University, India; Ajay Kumar Tiwari, UP Council of Sugarcane Research, India

                *Correspondence: Bhim P. Singh bhimpratap@ 123456gmail.com

                This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2016.00447
                4815437
                27065995
                Copyright © 2016 Leo, Passari, Joshi, Mishra, Uthandi, Ramesh, Gupta, Saikia, Sonawane and Singh.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Counts
                Figures: 8, Tables: 5, Equations: 0, References: 74, Pages: 13, Words: 9501
                Funding
                Funded by: Department of Biotechnology, Ministry of Science and Technology 10.13039/501100001407
                Award ID: BT/323/NE/TBP/2012
                Categories
                Microbiology
                Original Research

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