DOCSLIB.ORG

Community Structure Analysis 9

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Community Structure Analysis �9 Biodegradation of Polystyrene Foam by the Microorganisms from Landfill Pat Pataranutaporn ! Assistant prof. Savaporn Supaphol prof. Amornrat Phongdara Sureeporn Nualkaew Hi, I would like to invite you to take a look on my research Pat Introduction !3 “Styrofoam” Polystyrene Disadvantage Physical Properties ! • chemical formula is (C8H8)n • Non-biodegradable in the environment • monomer styrene • Made from non-renewable petroleum products • Thermoplastic • Chronic, low-level exposure risks undetermined • blowing agents Introduction !4 Bacteria nutritional requirements ! ‣ Energy source Biodegradation ‣ Carbon source Possibly work? ‣ Nitrogen source ‣ Minerals ‣ Water ‣ Growth factors Polystyrene structure http://faculty.ccbcmd.edu/courses/bio141/ lecguide/unit6/metabolism/growth/factors.html Introduction !5 Aims of the research ‣To identify the microbe that able to growth in the condition that polystyrene is a sole carbon source ! ‣To study the changing of microbe community structure in the selective culture which polystyrene is a sole carbon source ! ‣To observe the biodegradability of polystyrene To analyse the by product of polystyrene after degradation Methodology Methodology !7 Agar cultivation Community fingerprint 2 16s Ribosomal RNA Microbe Screening months later identification sampling Cultivation Molecular cloning Phylogenetic tree Degradability observation (SEM) Methodology Microbe sampling & cultivation !8 Agar cultivation Community fingerprint 2 16s Ribosomal RNA Microbe Screening months later identification sampling Cultivation Molecular cloning Phylogenetic tree Degradability observation (SEM) Methodology Community structure analysis !9 landfill that was contaminated by Polystyrene foam in Pattani,Thailand Styrofoam in the landfill Contaminated soil Methodology Community structure analysis !10 Sterile Polystyrene MSM broth ! ‣K2HPO4 ‣KH2PO4 ‣(NH4)2SO4 ‣MgSO4 ! ‣ FeSO4.2HO2 ‣MnCl2.4H2O ! ‣CoCl2.6H2O F S Control ‣CuCl2.2H2O ‣NiCl2.6H2O MSM broth MSM broth MSM broth ‣Na2MoO4.2H2O + Sterile Polystyrene + Sterile Polystyrene + Sterile Polystyrene ‣ZnSO4.7H2O + Landfill styrofoam + Landfill soil ‣H3BO3 Methodology Community structure analysis !11 Shake in shaker for 1 month then inoculate to new fresh broth for sub culture. Methodology Community structure analysis !12 Every week, The cell suspension in particular flask was taken to the eppendorf then stored at 2C๐ for stop bacteria growth. This solution used to monitor the changing of bacteria population. Methodology Community structure analysis !13 400 "l. 0 11 2 3 4 5 6 77 8 9 time(week) transfer culture Methodology Community structure analysis !14 Agar cultivation Community fingerprint 2 16s Ribosomal RNA Microbe Screening months later identification sampling Cultivation Molecular cloning Phylogenetic tree Degradability observation (SEM) Methodology Community structure analysis !15 Cell suspensions collected from each week of cultivation. DNA Extraction (Methode : QIAamp Protocol) 16s Ribosomal RNA Polymerase Chain Reaction (PCR) identification 16S rRNA gene Amplification Primer VFC &VR Denature Gradient Gel Electrophoresis (DGGE) Community Community structure analysis fingerprint Result Community structure analysis !16 DNA Replication : PCR(TopTaq Master Mix Kit) 16S rRNA gene Amplification by using Primer VR (Medlin et al., 1998) & VFC (Muyzer et al., 1993) Methodology Community structure analysis !17 Community structure trend time(week) Microbe diversity Dominant species Each DNA band represent 1 microbe DGGE Denature Gradient Gel Electrophoresis Looking for survivor! Result Community structure analysis !18 Bacteria from soil Bacteria from styrofoam Control DGGE 26/04/55 Marker Marker Negative Soil week 5 Soil week Soil week 6 Control week 6 Soil week 7 Soil week 8 Control week 7 Control week 8 Foam week 6 Foam week 7 Foam week 8 Foam week 5 week Foam Foam week 20 Soil week 1 Soil week Foam week 1 week Foam Marker Soil week 20 Running 300 minute from PCR product 24/04/55 template use 8 µl. Continuing band & found in control Continuing band Non-continuing band Molecular cloning & !19 Methodology identification Selected DNA Bacteria from soil Bacteria from styrofoam Control Marker Marker Marker Negative Soil week 5 Soil week Soil week 6 Control week 6 Soil week 7 Soil week 8 Control week 7 Control week 8 Foam week 6 Foam week 7 Foam week 8 Soil week 1 Soil week Foam week 5 week Foam Foam week 20 Foam week 1 week Foam Soil week 20 Selected for cloning Molecular cloning & !20 Methodology identification DNA from S week7, F week 7 and con week 7 Polymerase Chain Reaction (PCR) 16S rRNA gene Amplification Primer AF1 & 1541R 16s Ribosomal RNA identification Ligate with pGEM T-Easy Plasmid Transfer Plasmid to the competent cell (E.Coli) + Propagate extracted Plasmid + cutcheck with EcoR1 Molecular cloning Purify Plasmid + cutcheck with EcoR1 Nucleotide sequencing Blasting + Neighbourhood joining tree contracting Phylogenetic tree Molecular cloning & !21 Result identification PCR Product 16S rRNA gene Amplification extracted Plasmid + cutcheck with EcoR1 Primer AF1 & 1541R Gel electrophoresis Purify Plasmid + cutcheck with EcoR1 Molecular cloning & !22 Result identification Sequence report - Electropherogram F3 F10 S7 Control 4 Control 7 F5 Molecular cloning & !23 Result identification Sequence blasting Length Max Max Sample Similar sequence E.Value (bp) iden score F10 444 Herbasprillium.sp 98% 753 0.0 F3 504 Massialia aerilata 97% 830 0.0 S7 485 Caulobacter segnis ATCC 21756 98% 830 0.0 Control4 1,055 Azohydromonas australica 83% 1297 0.0 Control7 1,006 Ochrobactrum rhizosphaerea 82% 1193 0.0 Molecular cloning & !24 Result identification 0.00260 0.00130 Herbaspirillum chlorophenolicum 0.00260 0.00616 Herbaspirillum frisingense -0.00053 0.00390 Herbaspirillum seropedicae 0.00883 0.01006 F10 0.00952 -0.00301 Collimonas arenae 0.00479 Herminiimonas glaciei 0.00260 Janthinobacterium lividum 0.00561 0.01357 0.00000 0.00219 Janthinobacterium agaricidamnosum 0.00000 0.00260 Janthinobacterium agaricidamnosum(2) 0.02428 0.01535 Massilia brevitalea 0.00700 Naxibacter varians 0.00655 Naxibacter haematophilus 0.01396 0.05697 0.00523 0.00045 F3 0.00523 0.00132 Massilia aerilata 0.01596 Methylibium petroleiphilum PM1 Neighbourhood joining 0.01541 Schlegelella thermodepolymerans 0.02928 0.00521 tree contract from the 0.00511 Azohydromonas lata 0.00521 0.00055 Rubrivivax gelatinosus IL144 Specimen DNA sequence 0.00922 0.00509 Aquincola tertiaricarbonis 0.00391 0.51242 0.00110 Control4 0.00391 0.00531 Azohydromonas australica 0.00787 0.00541 Brevundimonas nasdae 0.00787 0.00023 Streptomyces longisporoflavus 0.01328 0.01434 Mycoplana bullata 0.00653 S7 0.02719 0.00653 0.00697 Caulobacter segnis ATCC 21756 0.02785 Phenylobacterium koreense 0.01553 0.04718 Rhizobium alamii 0.00260 0.01231 Ensifer adhaerens 0.03951 0.00260 Sinorhizobium fredii NGR234 0.01099 0.00061 Brucella ovis ATCC 25840 0.01259 Ochrobactrum haematophilum 0.00393 0.00783 -0.00160 Control7 0.00783 0.00476 Ochrobactrum rhizosphaerae out group 0.61463 Molecular cloning & !25 Result identification Found in soil Found in foam culture(F) Found in control ! culture(S) information Herbaspirillum Caulobacter segnis Massilia aerilata Ochrobactrum sp. Azohydromonas seropedicae Bacteria; Bacteria; Bacteria; Bacteria Bacteria;Proteobacte Proteobacteria; Proteobacteria; Proteobacteria; Proteobacteria riaBetaProteobacteri Alphaproteobacteria; Betaproteobacteria; Alphaproteobacteria; Betaproteobacteria Taxonomy a Burkholderiales Caulobacterales; Burkholderiales; Rhizobiales; Burkholderiales Oxalobacteraceae Caulobacteraceae; Oxalobacteraceae; Brucellaceae; Alcaligenaceae Massilia Caulobacter Herbaspirillum Ochrobactrum Azohydromonas Morphology & Negative, Bacilli, Negative, Bacilli, Negative, Spirilla, Negative, Bacilli Negative, Bacilli classification Aerobic, Mesophilic Aerobic Aerobic, Mesophilic Styrene ✓ Na ✓ ✓ Na degradation Aromatic ✓ compound ✓ Na ✓ Na degradation Carbon fixation - Na - ✓ Na Polycyclic aromatic ✓ Na - ✓ Na degradation Chlorophenol ✓ Na ✓ ✓ Na degradation Nitogen ✓ ✓ ✓ ✓ ✓ metabolism polyhydroxybutyrate (PHB) production Cellulose Other pathway ! degradation pathway Polyhydroxybutyrate fermentation Methodology Degradability Observation !26 Agar cultivation Community fingerprint 2 16s Ribosomal RNA Microbe Screening months later identification sampling Cultivation Molecular cloning Phylogenetic tree Degradability observation (SEM) Methodology Degradability Observation !27 The microscopic techniques ! ‣Test Method Used : In house method refer to WI-RES-SEM-Quanta-001 and WI-RES-SEM-001 ‣Test Equipment : Scanning Electron Microscope, Quanta40, FEI, Czech Republic ‣Test Technique : Electron micrograph ‣Test Condition Mode : low vacuum Detector : Large Field Detector()LFD High Voltage : 15.00,20.00 kV Methodology Degradability Observation !28 100x 200x 500x Control : Polystyrene in MSM broth without bacterial source. Regular polystyrene foam that didn’t use in experiment. Methodology Degradability Observation !29 100x 200x 500x Polystyrene in Medium with bacteria from Styrofoam in the landfill. Regular polystyrene foam that didn’t use in experiment. Methodology Degradability Observation !30 100x 200x 500x Polystyrene in Medium with bacteria from soil in the landfill. Regular polystyrene foam that didn’t use in experiment. Methodology Degradability Observation !31 Polystyrene in Medium with bacteria from soil in the landfill. Methodology Agar Cultivation !32 Agar cultivation Community fingerprint 2 16s Ribosomal RNA
Load more

Recommended publications
  • Changes in Acetylene Reduction Activities and Nifh Genes Associated with Field-Grown Sweet Potatoes with Different Nursery Farmers and Cultivars
    horticulturae Article Changes in Acetylene Reduction Activities and nifH Genes Associated with Field-Grown Sweet Potatoes with Different Nursery Farmers and Cultivars Kazuhito Itoh * , Keisuke Ohashi, Nao Yakai, Fumihiko Adachi and Shohei Hayashi Faculty of Life and Environmental Sciences, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan * Correspondence: [email protected]; Tel.: +81-852-32-6521 Received: 17 May 2019; Accepted: 25 July 2019; Published: 27 July 2019 Abstract: Sweet potato cultivars obtained from different nursery farmers were cultivated in an experimental field from seedling-stage to harvest, and the acetylene reduction activity (ARA) of different parts of the plant as well as the nifH genes associated with the sweet potatoes were examined. The relationship between these parameters and the plant weights, nitrogen contents, and natural abundance of 15N was also considered. The highest ARA was detected in the tubers and in September. Fragments of a single type of nitrogenase reductase gene (nifH) were amplified, and most of them had similarities with those of Enterobacteriaceae in γ-Proteobacteria. In sweet potatoes from one nursery farm, Dickeya nifH was predominantly detected in all of the cultivars throughout cultivation. In sweet potatoes from another farm, on the other hand, a transition to Klebsiella and Phytobacter nifH was observed after the seedling stage. The N2-fixing ability contributed to plant growth, and competition occurred between autochthonous and allochthonous bacterial communities in sweet potatoes. Keywords: sweet potato; endophyte; nitrogen fixation; nifH gene 1. Introduction The sweet potato (Ipomoea batatas L.) is a dicotyledonous plant that belongs to the family Convolvulaceae and is a subsistence crop with huge economic importance, especially in developing countries.
    [Show full text]
  • Linkages of Soil Nutrients and Diazotrophic Microbiome Under
    Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 October 2018 doi:10.20944/preprints201810.0382.v1 1 Article 2 Linkages of Soil Nutrients and Diazotrophic 3 Microbiome under Sugarcane-Legume Intercropping 4 Manoj Kumar Solanki1,4†, Chang-Ning Li1,2† Fei-Yong Wang1,2,3, Zhen Wang3, Tao-Ju Lan1, 5 Rajesh Kumar Singh1,3, Pratiksha Singh3, Li-Tao Yang3, Yang-Rui Li1, 2* 6 1 Guangxi Crop Genetic Improvement and Biotechnology Lab, Guangxi Academy of Agricultural Sciences, 7 Nanning 530007, Guangxi, China; [email protected] (M.K.S.), [email protected] (T.J.L.), 8 [email protected] (R.K.S.) 9 2 Guangxi Key Laboratory of Sugarcane Genetic Improvement, Key Laboratory of Sugarcane Biotechnology 10 and Genetic Improvement (Guangxi), Ministry of Agriculture, Sugarcane Research Institute, Guangxi 11 Academy of Agricultural Sciences, Sugarcane Research Center, Chinese Academy of Agricultural Sciences, 12 Nanning 530007, Guangxi, China; [email protected] (F.Y.W.), [email protected] (C.N.L.) 13 3 Agricultural College, State key Laboratory for Conservation and Utilization of Subtropical Agro- 14 bioresources, Guangxi University, Nanning 530004, Guangxi, China; [email protected] (Z.W.), 15 [email protected] (P.S.), [email protected] (L.T.Y.) 16 4 Agricultural Research Organization, Department of Postharvest and Food Sciences, The Volcani Center, 68 17 HaMaccabim Road, Rishon LeZion 7505101, Israel 18 19 *Yang-Rui Li, [email protected]; Tel: +86–771–3247689; Fax +86–771–3235318. 20 † These authors contributed equally to the work. 21 22 Abstract: Intercropping significantly improves land use efficiency and soil fertility. This study 23 examines the impact of three cultivation systems (monoculture sugarcane, peanut-sugarcane and 24 soybean-sugarcane intercropping) on soil properties and diazotrophs.
    [Show full text]
  • Appendix 1. Validly Published Names, Conserved and Rejected Names, And
    Appendix 1. Validly published names, conserved and rejected names, and taxonomic opinions cited in the International Journal of Systematic and Evolutionary Microbiology since publication of Volume 2 of the Second Edition of the Systematics* JEAN P. EUZÉBY New phyla Alteromonadales Bowman and McMeekin 2005, 2235VP – Valid publication: Validation List no. 106 – Effective publication: Names above the rank of class are not covered by the Rules of Bowman and McMeekin (2005) the Bacteriological Code (1990 Revision), and the names of phyla are not to be regarded as having been validly published. These Anaerolineales Yamada et al. 2006, 1338VP names are listed for completeness. Bdellovibrionales Garrity et al. 2006, 1VP – Valid publication: Lentisphaerae Cho et al. 2004 – Valid publication: Validation List Validation List no. 107 – Effective publication: Garrity et al. no. 98 – Effective publication: J.C. Cho et al. (2004) (2005xxxvi) Proteobacteria Garrity et al. 2005 – Valid publication: Validation Burkholderiales Garrity et al. 2006, 1VP – Valid publication: Vali- List no. 106 – Effective publication: Garrity et al. (2005i) dation List no. 107 – Effective publication: Garrity et al. (2005xxiii) New classes Caldilineales Yamada et al. 2006, 1339VP VP Alphaproteobacteria Garrity et al. 2006, 1 – Valid publication: Campylobacterales Garrity et al. 2006, 1VP – Valid publication: Validation List no. 107 – Effective publication: Garrity et al. Validation List no. 107 – Effective publication: Garrity et al. (2005xv) (2005xxxixi) VP Anaerolineae Yamada et al. 2006, 1336 Cardiobacteriales Garrity et al. 2005, 2235VP – Valid publica- Betaproteobacteria Garrity et al. 2006, 1VP – Valid publication: tion: Validation List no. 106 – Effective publication: Garrity Validation List no. 107 – Effective publication: Garrity et al.
    [Show full text]
  • Functional Metagenomics Using Pseudomonas Putida Expands the Known Diversity Of
    bioRxiv preprint doi: https://doi.org/10.1101/042705; this version posted March 10, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Functional metagenomics using Pseudomonas putida expands the known diversity of 2 polyhydroxyalkanoate synthases and enables the production of novel polyhydroxyalkanoate 3 copolymers 4 5 Jiujun Cheng and Trevor C. Charles* 6 7 Department of Biology and Centre for Bioengineering and Biotechnology, University of Waterloo, 8 Waterloo, Ontario, Canada N2L 3G1 9 10 *Corresponding author: 11 Dr. Trevor C. Charles 12 Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1. 13 E-mail: [email protected] 14 15 1 bioRxiv preprint doi: https://doi.org/10.1101/042705; this version posted March 10, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 16 17 Abstract 18 Bacterially produced biodegradable polyhydroxyalkanoates with versatile properties can be achieved 19 using different PHA synthase enzymes. This work aims to expand the diversity of known PHA 20 synthases via functional metagenomics, and demonstrates the use of these novel enzymes in PHA 21 production. Complementation of a PHA synthesis deficient Pseudomonas putida strain with a soil 22 metagenomic cosmid library retrieved 27 clones expressing either Class I, Class II or unclassified 23 PHA synthases, and many did not have close sequence matches to known PHA synthases.
    [Show full text]
  • Production and Chromatographic Analysis of Polyhydroxybutyrate from Waste Product Streams by Azohydromonas Lata
    Hochschule Rhein-Waal Rhine-Waal University of Applied Sciences Faculty of Life Sciences Production and Chromatographic Analysis of Polyhydroxybutyrate from waste product streams by Azohydromonas lata A Thesis Submitted in Partial Fulfilment of the Requirements of the Degree of Bachelor of Science in Bioengineering By Dominic Kösters Matriculation Number: 19056 Supervisors: Prof. Dr. Joachim Fensterle Prof. Dr. Peter Simon Submission Date: 04.11.2019 i Executive Summary As conventional plastic production receives much criticism for being harmful to both humans and the environment, this work focused on establishing a batch fermentation based on waste substrates from local industries to produce polyhydroxybutyrate (PHB) with Azohydromonas lata (A. lata). Additionally, the produced PHB was characterised by qualitative and quantitative gas chromatography coupled to a mass spectrometer (GC- MS) and gel permeation chromatography (GPC). Multiple substrates, including rhubarb waste, pea waste, and dairy industry wastewater (KAAS medium), were tested for successful fermentation. Rhubarb and pea waste could not be used as a fermentation substrate due to obstacles in upstream processing. KAAS medium was prepared by centrifugation and sterile filtration and tested in a 1 L and 4 L fermentation yielding non- competitive biomass and PHB concentrations with 0.09 g/L and 0.004 g/L, respectively. A 3 L fermentation with pure saccharose as the carbon source served as a model for comparison and analysis and resulted in a measured biomass concentration of 3.58 g/L with a PHB content of 1.43 g/L after Soxhlet extraction. The GPC analysis led to no conclusive elograms and results due to issues in non-destructively extracting and solving the PHB.
    [Show full text]
  • Évaluation Des Risques Sanitaires Liés À L'exposition Par Ingestion
    Évaluation des risques sanitaires liés à l’exposition par ingestion de Pseudomonades dans les eaux destinées à la consommation humaine (hors eaux conditionnées) Rapport Octobre 2010 Édition scientifique Évaluation des risques sanitaires liés à l’exposition par ingestion de Pseudomonades dans les eaux destinées à la consommation humaine (hors eaux conditionnées) Rapport Octobre 2010 Édition scientifique Sommaire COMPOSITION DU GROUPE DE TRAVAIL……………………………………………………….………..5 LISTE DES SIGLES ET ACRONYMES……………………………………………………………………….6 GLOSSAIRE………………………………………………………………………………………..….…………7 I. INTRODUCTION…………………………………………………………………………….………..…....9 II. PSEUDOMONADES: GÉNÉRALITÉS ......................................................................................... 11 1. Taxonomie : état des lieux (octobre 2009) 2. Caractéristiques des Pseudomonades 3. Écologie bactérienne 3.1. Habitat .................................................................................................................................... 12 3.2. Les biofilms ............................................................................................................................. 13 III. SURVIE ET CROISSANCE DES PSEUDOMONADES DANS LES EAUX ................................. 14 1. Survie et croissance dans les eaux 1.1. Eaux conditionnées ................................................................................................................ 14 1.2. Eau de distribution publique (EDP) .......................................................................................
    [Show full text]
  • 1 Genomic Evidence for the Degradation of Terrestrial Organic Matter by Pelagic Arctic
    bioRxiv preprint doi: https://doi.org/10.1101/325027; this version posted May 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Genomic evidence for the degradation of terrestrial organic matter by pelagic Arctic 2 Ocean Chloroflexi bacteria 3 4 David Colatriano1, Patricia Tran1, Celine Guéguen2, William J. Williams3, Connie Lovejoy4, 5, and 5 David A. Walsh1* 6 7 1 Department of Biology, Concordia University, 7141 Sherbrooke St. West, Montreal, 8 Quebec,H4B 1R6, Canada 9 2 Department of Chemistry and School of the Environment, Trent University, 1600 West bank 10 Drive, Peterborough, Ontario, K9J 7B8, Canada 11 3 Fisheries and Oceans Canada, Institute of Ocean Sciences, 9860 West Saanich Road, 12 Sidney, British Columbia, V8V 4L1, Canada 13 4 Département de biologie, Institut de Biologie Intégrative et des Systèmes (IBIS)and Québec- 14 Océan, Université Laval, Québec, G1K 7P4, Canada 15 5.Takuvik Joint International Laboratory (UMI 3376), Université Laval (Canada) - CNRS 16 (France), Université Laval, Québec QC G1V 0A6, Canada 17 18 19 *Corresponding author: Phone: (514) 848-2424 (ext. 3477) 20 E-mail: [email protected] 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/325027; this version posted May 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 Abstract 25 The Arctic Ocean currently receives a large supply of global river discharge and terrestrial 26 dissolved organic matter.
    [Show full text]
  • Expressed Nifh Genes of Endophytic Bacteria Detected in Field-Grown Sweet Potatoes (Ipomoea Batatas L.)
    Microbes Environ. Vol. 23, No. 1, 89–93, 2008 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.23.89 Expressed nifH Genes of Endophytic Bacteria Detected in Field-Grown Sweet Potatoes (Ipomoea batatas L.) JUNKO TERAKADO-TONOOKA1,2*, YOSHINARI OHWAKI1, HIROMOTO YAMAKAWA3, FUKUYO TANAKA1, TADAKATSU YONEYAMA4, and SHINSUKE FUJIHARA1 1National Agricultural Research Center, Kannondai 3–1–1, Tsukuba, Ibaraki 305–8666, Japan; 2JSPS Research Fellow, Japan Society for the Promotion of Science, Ichi-ban-cho 8, Chiyoda-ku, Tokyo 102–8472, Japan; 3National Agricultural Research Center, Hokuriku Research Center, Inada 1–2–1, Jyoetsu, Nigata 943–0193, Japan; and 4Department of Applied Biological Chemistry, University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113–8657, Japan (Received November 2, 2007—Accepted December 27, 2007) We examined the nitrogenase reductase (nifH) genes of endophytic diazotrophic bacteria expressed in field-grown sweet potatoes (Ipomoea batatas L.) by reverse transcription (RT)-PCR. Gene fragments corresponding to nifH were amplified from mRNA obtained from the stems and storage roots of field-grown sweet potatoes several months after planting. Sequence analysis revealed that these clones were homologous to the nifH sequences of Bradyrhizobium, Pelomonas, and Bacillus sp. in the DNA database. Investigation of the nifH genes amplified from the genomic DNA extracted from these sweet potatoes also showed high similarity to various α-proteobacteria including Bradyrhizobium, β-proteobacteria, and cyanobacteria. These results suggest that bradyrhizobia colonize and express nifH genes not only in the root nodules of leguminous plants but also in sweet potatoes as diazotrophic endophytes. Key words: endophyte, sweet potato, nitrogen fixation, nifH, RT-PCR The sweet potato (Ipomoea batatas L.) is known for its identify active diazotrophic bacteria, we examined the ability to grow well in nitrogen (N)-poor soils16).
    [Show full text]
  • Non -Supplemented’ Represents the Unamended Sediment
    Figure S1: Control microcosms for the ibuprofen degradation experiment (Fig. 1). ‘Non -supplemented’ represents the unamended sediment. The abiotic controls ‘Sorption’ and ‘Hydrolysis’ contained autoclaved sediment and river water, respectively, and were amended with 200 µM ibuprofen. Value s are the arithmetic means of three replicate incubations. Red and blue-headed arrows indicate sampling of the sediment for nucleic acids extraction after third and fifth re-spiking, respectively. Error bars indicating standard deviations are smaller than the size of the symbols and therefore not apparent. Transformation products Transformation Transformation products Figure S3: Copy numbers of the 16S rRNA gene and 16S rRNA detected in total bacterial community. Sample code: A, amended with 1 mM acetate and ibuprofen per feeding; 0, 5, 40, 200, and 400, indicate supplemental ibuprofen concentrations of 0 µM, 5 µM,40 µM, 200 µM and 400 µM, respectively, given per feeding; 0’, 3’, and 5’, correspond to samples obtained at the start of the incubation, and after the third and fifth refeeding, respectively. Sampling times for unamended controls were according to those of the 400 µM ibuprofen treatment. Values are the arithmetic average of three replicates. Error bars indicate standard deviation values. Figure S4: Alpha diversity and richness estimators of 16S rRNA gene and 16S rRNA obtained from Illumina amplicon sequencing. Sample code: A, amended with 1 mM acetate and ibuprofen per feeding; 0, 5, 40, 200, and 400, indicate supplemental ibuprofen concentrations of 0 µM, 5 µM, 40 µM, 200 µM and 400 µM, respectively, given per feeding; 0’, 3’, and 5’, correspond to samples obtained at the start of the incubation, and after the third and fifth refeeding, respectively.
    [Show full text]
  • Development of a PCR-Based Method for Monitoring the Status of Alcaligenes Species in the Agricultural Environment
    Biocontrol Science, 2014, Vol. 19, No. 1, 23－31 Original Development of a PCR-Based Method for Monitoring the Status of Alcaligenes Species in the Agricultural Environment MIYO NAKANO1, MASUMI NIWA2, AND NORIHIRO NISHIMURA1＊ 1 Department of Translational Medical Science and Molecular and Cellular Pharmacology, Pharmacogenomics, and Pharmacoinformatics, Mie University Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie 514-8507, Japan 2 DESIGNER FOODS. Co., Ltd. NALIC207, Chikusa 2-22-8, Chikusa-ku, Nagoya, Aichi 464-0858, Japan Received 1 April, 2013/Accepted 14 September, 2013 To analyze the status of the genus Alcaligenes in the agricultural environment, we developed a PCR method for detection of these species from vegetables and farming soil. The selected PCR primers amplified a 107-bp fragment of the 16S rRNA gene in a specific PCR assay with a detection limit of 1.06 pg of pure culture DNA, corresponding to DNA extracted from approxi- mately 23 cells of Alcaligenes faecalis. Meanwhile, PCR primers generated a detectable amount of the amplicon from 2.2×102 CFU/ml cell suspensions from the soil. Analysis of vegetable phyl- loepiphytic and farming soil microbes showed that bacterial species belonging to the genus Alcaligenes were present in the range from 0.9×100 CFU per gram（ or cm2）（ Japanese radish: Raphanus sativus var. longipinnatus） to more than 1.1×104 CFU/g（ broccoli flowers: Brassica oleracea var. italic）, while 2.4×102 to 4.4×103 CFU/g were detected from all soil samples. These results indicated that Alcaligenes species are present in the phytosphere at levels 10–1000 times lower than those in soil.
    [Show full text]
  • Outline Release 7 7C
    Taxonomic Outline of Bacteria and Archaea, Release 7.7 Taxonomic Outline of the Bacteria and Archaea, Release 7.7 March 6, 2007. Part 4 – The Bacteria: Phylum “Proteobacteria”, Class Betaproteobacteria George M. Garrity, Timothy G. Lilburn, James R. Cole, Scott H. Harrison, Jean Euzéby, and Brian J. Tindall Class Betaproteobacteria VP Garrity et al 2006. N4Lid DOI: 10.1601/nm.16162 Order Burkholderiales VP Garrity et al 2006. N4Lid DOI: 10.1601/nm.1617 Family Burkholderiaceae VP Garrity et al 2006. N4Lid DOI: 10.1601/nm.1618 Genus Burkholderia VP Yabuuchi et al. 1993. GOLD ID: Gi01836. GCAT ID: 001596_GCAT. Sequenced strain: SRMrh-20 is from a non-type strain. Genome sequencing is incomplete. Number of genomes of this species sequenced 2 (GOLD) 1 (NCBI). N4Lid DOI: 10.1601/nm.1619 Burkholderia cepacia VP (Palleroni and Holmes 1981) Yabuuchi et al. 1993. <== Pseudomonas cepacia (basonym). Synonym links through N4Lid: 10.1601/ex.2584. Source of type material recommended for DOE sponsored genome sequencing by the JGI: ATCC 25416. High-quality 16S rRNA sequence S000438917 (RDP), U96927 (Genbank). GOLD ID: Gc00309. GCAT ID: 000301_GCAT. Entrez genome id: 10695. Sequenced strain: ATCC 17760, LMG 6991, NCIMB9086 is from a non-type strain. Genome sequencing is completed. Number of genomes of this species sequenced 1 (GOLD) 1 (NCBI). N4Lid DOI: 10.1601/nm.1620 Pseudomonas cepacia VP (ex Burkholder 1950) Palleroni and Holmes 1981. ==> Burkholderia cepacia (new combination). Synonym links through N4Lid: 10.1601/ex.2584. Source of type material recommended for DOE sponsored genome sequencing by the JGI: ATCC 25416. High- quality 16S rRNA sequence S000438917 (RDP), U96927 (Genbank).
    [Show full text]
  • Applied T-Rflp Analyses for the Identification And
    APPLIED T-RFLP ANALYSES FOR THE IDENTIFICATION AND CHARACTERIZATION OF MICROBIAL POPULATIONS ASSOCIATED WITH DAMPING-OFF INCIDENCE IN A TRANSITIONAL ORGANIC CROPPING SYSTEM DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By María Soledad Benítez, M.S. * * * The Ohio State University 2008 Dissertation Committee: Approved by: Dr. Brian McSpadden Gardener, Adviser Dr. Terrence L. Graham ___________________________ Dr. Warren Dick Adviser Dr. Enrico Bonello Plant Pathology Graduate Program ABSTRACT Within agricultural ecosystems the abundance and activities of diverse microbial populations affecting plant health can be significantly influenced by farm management practices. This work describes the effects of transitional cropping strategies on damping- off incidence and on soil and rhizosphere bacterial, fungal and oomycete communities. Specifically, individual microbial populations were associated with damping-off incidence and the suppression thereof, and two novel bacterial species were characterized for their ability to reduce seedling disease severity. Soils from an organic transition experiment exhibited different levels of damping-off suppression, with a perennial mixed hay transition consistently suppressing damping-off of tomato and soybean. Microbial community analyses were performed from the rhizosphere and surrounding soil of tomato and soybean seedlings grown in soils previously exposed to different transition strategies. The abundance of Pseudomonas and DAPG-producing Pseudomonas populations, did not clearly associate with disease levels. However, the analysis of bacterial, fungal and oomycete community profiles (generated from terminal restriction fragment length polymorphism of 16S and ITS genes, respectively) revealed individual members of the community (expressed as a terminal restriction fragment, TRF) that statistically associated with disease suppression or disease.
    [Show full text]