<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> <html> <head> <title>UTas ePrints - Bacterial community shifts in organically perturbed sediments</title> <script type="text/javascript" src="http://eprints.utas.edu.au/javascript/auto.js"><!-- padder --></script> <style type="text/css" media="screen">@import url(http://eprints.utas.edu.au/style/auto.css);</style> <style type="text/css" media="print">@import url(http://eprints.utas.edu.au/style/print.css);</style> <link rel="icon" href="/images/eprints/favicon.ico" type="image/x-icon" /> <link rel="shortcut icon" href="/images/eprints/favicon.ico" type="image/x-icon" /> <link rel="Top" href="http://eprints.utas.edu.au/" /> <link rel="Search" href="http://eprints.utas.edu.au/cgi/search" /> <meta content="Bissett, Andrew" name="eprints.creators_name" /> <meta content="Burke, Chris" name="eprints.creators_name" /> <meta content="Cook, Perran L.M." name="eprints.creators_name" /> <meta content="Bowman, John P." name="eprints.creators_name" /> <meta content="abissett@mpi-bremen.de" name="eprints.creators_id" /> <meta content="C.Burke@utas.edu.au" name="eprints.creators_id" /> <meta name="eprints.creators_id" /> <meta content="john.bowman@utas.edu.au" name="eprints.creators_id" /> <meta content="article" name="eprints.type" /> <meta content="2007-09-12" name="eprints.datestamp" /> <meta content="2008-02-08T05:03:57Z" name="eprints.lastmod" /> <meta content="show" name="eprints.metadata_visibility" /> <meta content="Bacterial community shifts in organically perturbed sediments" name="eprints.title" /> <meta content="pub" name="eprints.ispublished" /> <meta content="270702" name="eprints.subjects" /> <meta content="300703" name="eprints.subjects" /> <meta content="270307" name="eprints.subjects" /> <meta content="restricted" name="eprints.full_text_status" /> <meta content="sediment microbiology, fish farm" name="eprints.keywords" /> <meta content="The definitive version is available at www.blackwell-synergy.com" name="eprints.note" /> <meta content="Bacterial abundance, diversity and sediment function were investigated in organically perturbed sediments under Tasmanian salmon (Salmo salar) farms and adjacent reference sites. Bacterial numbers increased as farming and organic loading progressed through the farm stocking cycle and declined during the fallow period, although not to prestocking levels. Bacterial numbers ranged between approximately 2 x 10 to the power of 8 and 3 x 10 to the power of 9 cells per gram of sediment and were higher at cage sites than reference sites. Microelectrode and respiration data also demonstrated a clear effect of organic loading on sediments. Denaturing gradient gel electrophoresis (DGGE) showed that bacterial communities shifted both in response to farm loading and its cessation. A seasonal effect on microbial communities was also evident. Although bacterial communities did shift again during the fallowing period, this shift was not necessarily a return to preloading communities. The complexity of community shifts may be affected by the vast functional redundancy of bacterial groups. All bacterial communities, including those at reference sites, were highly dynamic. Respiration studies of amended sediments indicated that fish farm sediments were at least as resilient and diverse as reference site communities. The results of this study indicate that the functional redundancy of highly complex bacterial communities contributes to their robustness. The relationship between diversity and stability in bacterial communities remains unclear and requires further investigation before an understanding of bacterial response to perturbation is possible." name="eprints.abstract" /> <meta content="2007-01" name="eprints.date" /> <meta content="submitted" name="eprints.date_type" /> <meta content="Environmental Microbiology" name="eprints.publication" /> <meta content="9" name="eprints.volume" /> <meta content="1" name="eprints.number" /> <meta content="46-60" name="eprints.pagerange" /> <meta content="10.1111/j.1462-2920.2006.01110.x" name="eprints.id_number" /> <meta content="UNSPECIFIED" name="eprints.thesis_type" /> <meta content="TRUE" name="eprints.refereed" /> <meta content="1462-2920" name="eprints.issn" /> <meta content="http://dx.doi.org/10.1111/j.1462-2920.2006.01110.x" name="eprints.official_url" /> <meta content="Almgren, T., Dryssen, D., and Fonselius, S. (1983) Determination of alkalinity and total carbonate. In Methods of Seawater Analysis. Kremling, K. (eds). Weinheim, Germany: Verlag Chemie, pp. 99-123. ¢Anderson, M.J. (2003a) NPMANOVA: a FORTRAN computer program for non-parametric multivariate analysis of variance (for any two factor design) using permutation tests. Auckland, New Zealand: Department of Statistics, University of Auckland. http://www.stat.auckland.ac.nz/~mja/Programs.htm. Anderson, M.J. (2003b) xmatrix: a FORTRAN computer program for calculating design matrices for terms in ANOVA designs in a linear model. Auckland, New Zealand: Department of Statistics, University of Auckland. http://www.stat.auckland.ac.nz/~mja/Programs.htm. Anderson, M.J. (2003c) distlm v.2: a FORTRAN computer program to calculate a distance-based multivariate analysis for a linear model. Auckland, New Zealand: Department of Statistics, University of Auckland. http://www.stat.auckland.ac.nz/~mja/Programs.htm. Anderson, M.J. (2003d) CAP: a FORTRAN computer program for canonical analysis of principle cooradinates. Auckland, New Zealand: Department of Statistics, University of Auckland. http://www.stat.auckland.ac.nz/~mja/Programs.htm. Bissett, A., Bowman, J.P., and Burke, C. (2006) Bacterial diversity in organically-enriched fish farm sediments. FEMS Microbiol Ecol 55: 48-56. Synergy, Medline, ISI Christensen, P.B., Rysgaard, S., Sloth, N.P., Dalsgaard, T., and Schwaerter, S. (2000) Sediment mineralization, nutrient fluxes, denitrification and dissimilatory nitrate reduction to ammonium in an estuarine fjord with sea cage trout farms. Aquat Microbial Ecol 21: 73-84. CrossRef, ISI Clarke, K.R., and Warwick, R.M. (2001) Change in Marine Communities: an Approach to Statistical Analysis and Interpretation. Plymouth, UK: PRIMER-E. Dalsgaard, T., Nielsen, L.P., Brotas, V., Viaroli, P., Underwood, G., et al. (2000) Protocol Handbook for NICE - Nitrogen Cycling in Estuaries: A Project Under the EU Research Programme: Marine Science and Technology MAST III. Silkeborg, Denmark: National Environmental Research Institute. Epstein, S.S., and Rossel, J. (1995) Enumeration of sandy sediment bacteria search for optimal protocol. Mar Ecol Prog Series 117: 289-298. ISI, CSA Felsing, M., Glenncross, B., and Telfer, T.C. (2005) Preliminary study on the effects of exclusion of wild fauna from aquaculture cages in a shallow marine environment. Aquaculture 243: 159-174. CrossRef, ISI Ferris, M., Muyzer, G., and Ward, D. (1996) Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol 62: 340-346. Medline, ISI, CSA Findlay, R., Watling, L., and Mayer, L. (1995) Environmental impact of salmon net-pen culture on marine benthic communities in Maine: a case study. Estuaries 18: 145-179. CrossRef, ISI, CSA Girvan, M.S., Campbell, C.D., Killham, K., Prosser, J.I., and Glover, L.A. (2005) Bacterial diversity promotes community stability and functional resilience after perturbation. Environ Microbiol 7: 301-313. Synergy, Medline, ISI, Chemport Gough, H.L., and Stahl, D. (2003) Optimization of direct cell counting in sediment. J Microbiol Meth 52: 39-46. CrossRef, Medline, ISI, CSA Heip, C.H.R., Goosen, N.K., Herman, P.M.J., Kromkamp, J., Middelburg, J.J., and Soetaert, K. (1995) Production and consumption of biological particles in temperate tidal estuaries. Oceanography and Marine Biology - an Annual Review, 33, 1-149. ISI, CSA La Rosa, T., Mirto, S., Marino, A., Alonzo, V., Maugeri, T.L., and Mazzola, A. (2001) Heterotrophic bacteria community and pollution indicators of mussel-farm impact in the gulf of Gaeta (Tyrrhenian Sea). Mar Environ Res 52: 301-321. CrossRef, Medline, ISI, CSA La Rosa, T., Mirto, S., Mazzola, A., and Maugeri, T.L. (2004) Benthic microbial indicators of fish farm impact in a coastal area of the Tyrrhenian Sea. Aquaculture 230: 153-167. CrossRef, ISI Lane, D.J. (1991) 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics. Stackbrandt, E., and Goodfellow, M. (eds). New York, USA: John Wiley and Sons, pp. 115-175. Langenheder, S., Lindstrom, E.S., and Tranvik, L.J. (2006) Structure and function of bacterial communities emerging from different sources under identical conditions. Appl Environ Microbiol 72: 212-220. CrossRef, Medline, ISI Legendre, P., and Anderson, M.J. (1999) Distance-based redundancy analysis: testing multispecies responses in multifactorial ecological experiments. Ecol Monographs 69: 1-24. CrossRef, ISI, CSA Macleod, C.K., Crawford, C.M., and Moltschaniwskyj, N.A. (2004) Assessment of long term change in sediment condition after organic enrichment: defining recovery. Marine Pollution Bulletin 49: 79-88. CrossRef, Medline, ISI Middelburg, J.J., Duarte, C.M., and Gattuso, J.P. (2005) Respiration in coastal benthic communities. In Respiration in Aquatic Ecosystems. Williams, P.J. (eds). Oxford, UK: Oxford University Press, pp. 206 -224. Morris, C.E., Bardin, M., Berg, O., Frey-Klett, P., Fromin, N., Girardin, H., et al. (2002) Microbial biodiversity: approaches to experimental design and hypothesis testing in primary scientific literature from 1975 to 1999. Microbiol Mol Biol Rev 66: 592-616. CrossRef, Medline, ISI, CSA Muller, A.K., Westergaard, K., Christensen, S., and Sorensen, S.J. (2002) The diversity and function of soil microbial communities exposed to different disturbances. Microbial Ecol 44: 49-58. CrossRef, Medline, ISI, CSA Noble, R.T., and Fuhrman, J.A. (1998) Use of Sybr Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquat Microbial Ecol 14: 113-118. ISI, CSA Nordgren, A., Baath, E., and Soderstrom, B. (1988) Evaluation of soil respiration characteristics to assess heavy metal effects on soil microorganisms using glutamic acid as a substrate. Soil Biol Biochem 20: 949-954. CrossRef, ISI, CSA Paerl, H.W. (1998) Structure and function of anthropogenically altered microbial communities in coastal waters [Review]. Current Opinion Microbiol 1: 296-302. CrossRef, Medline, ISI, CSA Pearson, T.H., and Rosenberg, R. (1978) Macrobenthic succession in relation to organic enrichment and pollution of the marine environment. In Oceanography and Marine Biology: an Annual Review. Barnes, M., (ed.). Aberdeen, UK: Aberdeen University Press, 16: pp. 229-311. Rasmussen, H., and Jorgensen, B.B. (1992) Microelectrode studies of seasonal oxygen-uptake in a coastal sediment- role of molecular-diffusion. Mar Ecol Prog Ser 81: 289-303. ISI, CSA Revsbech, N.P. (1989) An oxygen microsensor with a guard cathode. Limnol Oceanogr 34: 474. ISI, Chemport, CSA Revsbech, N.P., and Jorgensen, B.B. (1986) Microelectrodes: their use in microbial ecology. Adv Microb Ecol 9: 293-352. ISI Roy, R.N., Roy, L.N., Vogel, K.M., Porter-Moore, K.M., Pearson, T., Good, C.E., et al. (1993) The dissociation constants of carbonic acid in seawater at salinities 5 -45 and temperatures 0 -45°C. Mar Chem 44: 249-267. CrossRef, ISI, CSA Rysgaard, S., Thamdrup, B., Risgaardpetersen, N., Fossing, H., Berg, P., Bondo Christensen, P., and Dalsgaard, T. (1998) Seasonal carbon and nutrient mineralization in high-Arctic coastal marine sediment, Young Sound, Northeast Greenland. Mar Ecol Prog Series 175: 261-276. ISI, CSA Savage, C., Elmgren, R., and Larsson, U. (2002) Effects of sewerage-derived nutrients on an estuarine macrobenthic community. Mar Ecol Prog Series 243: 67-82. CrossRef, ISI, CSA Torsvik, V., Sorheim, R., and Goksoyr, J. (1996) Total bacterial diversity in soil and sediment communities - a review [Review]. J Ind Microbiol 17: 170-178. CrossRef Vezzulli, L., Chelossi, E., Riccardi, G., and Fabiano, M. (2002) Bacterial community structure and activity in fish farm sediments of the Ligurian Sea (Western Mediterranean). Aquac Int 10: 123-141. CrossRef, ISI Viles, C., and Sieracki, M. (1992) Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy. Appl Environ Microbiol 58: 584-592. Medline, ISI, Chemport, CSA Wildish, D.J., Hargrave, B.T., and Pohle, G. (2001)Cost-effective monitoring of organic enrichment resulting from salmon mariculture. ICES J Mar Sci 58: 469-476. CrossRef, ISI, CSA " name="eprints.referencetext" /> <meta content="Bissett, Andrew and Burke, Chris and Cook, Perran L.M. and Bowman, John P. (2007) Bacterial community shifts in organically perturbed sediments. Environmental Microbiology, 9 (1). pp. 46-60. ISSN 1462-2920" name="eprints.citation" /> <meta content="http://eprints.utas.edu.au/1902/1/j.1462-2920.2006.01110.pdf" name="eprints.document_url" /> <link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /> <meta content="Bacterial community shifts in organically perturbed sediments" name="DC.title" /> <meta content="Bissett, Andrew" name="DC.creator" /> <meta content="Burke, Chris" name="DC.creator" /> <meta content="Cook, Perran L.M." name="DC.creator" /> <meta content="Bowman, John P." name="DC.creator" /> <meta content="270702 Marine and Estuarine Ecology (incl. Marine Ichthyology)" name="DC.subject" /> <meta content="300703 Aquaculture" name="DC.subject" /> <meta content="270307 Microbial Ecology" name="DC.subject" /> <meta content="Bacterial abundance, diversity and sediment function were investigated in organically perturbed sediments under Tasmanian salmon (Salmo salar) farms and adjacent reference sites. Bacterial numbers increased as farming and organic loading progressed through the farm stocking cycle and declined during the fallow period, although not to prestocking levels. Bacterial numbers ranged between approximately 2 x 10 to the power of 8 and 3 x 10 to the power of 9 cells per gram of sediment and were higher at cage sites than reference sites. Microelectrode and respiration data also demonstrated a clear effect of organic loading on sediments. Denaturing gradient gel electrophoresis (DGGE) showed that bacterial communities shifted both in response to farm loading and its cessation. A seasonal effect on microbial communities was also evident. Although bacterial communities did shift again during the fallowing period, this shift was not necessarily a return to preloading communities. The complexity of community shifts may be affected by the vast functional redundancy of bacterial groups. All bacterial communities, including those at reference sites, were highly dynamic. Respiration studies of amended sediments indicated that fish farm sediments were at least as resilient and diverse as reference site communities. The results of this study indicate that the functional redundancy of highly complex bacterial communities contributes to their robustness. The relationship between diversity and stability in bacterial communities remains unclear and requires further investigation before an understanding of bacterial response to perturbation is possible." name="DC.description" /> <meta content="2007-01" name="DC.date" /> <meta content="Article" name="DC.type" /> <meta content="PeerReviewed" name="DC.type" /> <meta content="application/pdf" name="DC.format" /> <meta content="http://eprints.utas.edu.au/1902/1/j.1462-2920.2006.01110.pdf" name="DC.identifier" /> <meta content="http://dx.doi.org/10.1111/j.1462-2920.2006.01110.x" name="DC.relation" /> <meta content="Bissett, Andrew and Burke, Chris and Cook, Perran L.M. and Bowman, John P. 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border: solid 1px #ccc; padding: 3px"><tr> <td align="left"><a href="http://eprints.utas.edu.au/cgi/users/home">Login</a> | <a href="http://eprints.utas.edu.au/cgi/register">Create Account</a></td> <td align="right" style="white-space: nowrap"> <form method="get" accept-charset="utf-8" action="http://eprints.utas.edu.au/cgi/search" style="display:inline"> <input class="ep_tm_searchbarbox" size="20" type="text" name="q" /> <input class="ep_tm_searchbarbutton" value="Search" type="submit" name="_action_search" /> <input type="hidden" name="_order" value="bytitle" /> <input type="hidden" name="basic_srchtype" value="ALL" /> <input type="hidden" name="_satisfyall" value="ALL" /> </form> </td> </tr></table></td></tr> <tr> <td class="toplinks"><!-- InstanceBeginEditable name="content" --> <div align="center"> <table width="720" class="ep_tm_main"><tr><td align="left"> <h1 class="ep_tm_pagetitle">Bacterial community shifts in organically perturbed sediments</h1> <p style="margin-bottom: 1em" class="not_ep_block"><span class="person_name">Bissett, Andrew</span> and <span class="person_name">Burke, Chris</span> and <span class="person_name">Cook, Perran L.M.</span> and <span class="person_name">Bowman, John P.</span> (2007) <xhtml:em>Bacterial community shifts in organically perturbed sediments.</xhtml:em> Environmental Microbiology, 9 (1). pp. 46-60. ISSN 1462-2920</p><p style="margin-bottom: 1em" class="not_ep_block"></p><table style="margin-bottom: 1em" class="not_ep_block"><tr><td valign="top" style="text-align:center"><a href="http://eprints.utas.edu.au/1902/1/j.1462-2920.2006.01110.pdf"><img alt="[img]" src="http://eprints.utas.edu.au/style/images/fileicons/application_pdf.png" class="ep_doc_icon" border="0" /></a></td><td valign="top"><a href="http://eprints.utas.edu.au/1902/1/j.1462-2920.2006.01110.pdf"><span class="ep_document_citation">PDF</span></a> - Full text restricted - Requires a PDF viewer<br />255Kb</td><td><form method="get" accept-charset="utf-8" action="http://eprints.utas.edu.au/cgi/request_doc"><input accept-charset="utf-8" value="2397" name="docid" type="hidden" /><div class=""><input value="Request a copy" name="_action_null" class="ep_form_action_button" onclick="return EPJS_button_pushed( '_action_null' )" type="submit" /> </div></form></td></tr></table><p style="margin-bottom: 1em" class="not_ep_block">Official URL: <a href="http://dx.doi.org/10.1111/j.1462-2920.2006.01110.x">http://dx.doi.org/10.1111/j.1462-2920.2006.01110.x</a></p><div class="not_ep_block"><h2>Abstract</h2><p style="padding-bottom: 16px; text-align: left; margin: 1em auto 0em auto">Bacterial abundance, diversity and sediment function were investigated in organically perturbed sediments under Tasmanian salmon (Salmo salar) farms and adjacent reference sites. Bacterial numbers increased as farming and organic loading progressed through the farm stocking cycle and declined during the fallow period, although not to prestocking levels. Bacterial numbers ranged between approximately 2 x 10 to the power of 8 and 3 x 10 to the power of 9 cells per gram of sediment and were higher at cage sites than reference sites. Microelectrode and respiration data also demonstrated a clear effect of organic loading on sediments. Denaturing gradient gel electrophoresis (DGGE) showed that bacterial communities shifted both in response to farm loading and its cessation. A seasonal effect on microbial communities was also evident. Although bacterial communities did shift again during the fallowing period, this shift was not necessarily a return to preloading communities. The complexity of community shifts may be affected by the vast functional redundancy of bacterial groups. All bacterial communities, including those at reference sites, were highly dynamic. Respiration studies of amended sediments indicated that fish farm sediments were at least as resilient and diverse as reference site communities. The results of this study indicate that the functional redundancy of highly complex bacterial communities contributes to their robustness. The relationship between diversity and stability in bacterial communities remains unclear and requires further investigation before an understanding of bacterial response to perturbation is possible.</p></div><table style="margin-bottom: 1em" cellpadding="3" class="not_ep_block" border="0"><tr><th valign="top" class="ep_row">Item Type:</th><td valign="top" class="ep_row">Article</td></tr><tr><th valign="top" class="ep_row">Additional Information:</th><td valign="top" class="ep_row">The definitive version is available at www.blackwell-synergy.com</td></tr><tr><th valign="top" class="ep_row">Keywords:</th><td valign="top" class="ep_row">sediment microbiology, fish farm</td></tr><tr><th valign="top" class="ep_row">Subjects:</th><td valign="top" class="ep_row"><a href="http://eprints.utas.edu.au/view/subjects/270702.html">270000 Biological Sciences > 270700 Ecology and Evolution > 270702 Marine and Estuarine Ecology (incl. Marine Ichthyology)</a><br /><a href="http://eprints.utas.edu.au/view/subjects/300703.html">300000 Agricultural, Veterinary and Environmental Sciences > 300700 Fisheries Sciences > 300703 Aquaculture</a><br /><a href="http://eprints.utas.edu.au/view/subjects/270307.html">270000 Biological Sciences > 270300 Microbiology > 270307 Microbial Ecology</a></td></tr><tr><th valign="top" class="ep_row">Collections:</th><td valign="top" class="ep_row">UNSPECIFIED</td></tr><tr><th valign="top" class="ep_row">ID Code:</th><td valign="top" class="ep_row">1902</td></tr><tr><th valign="top" class="ep_row">Deposited By:</th><td valign="top" class="ep_row"><span class="ep_name_citation"><span class="person_name">Dr Chris Burke</span></span></td></tr><tr><th valign="top" class="ep_row">Deposited On:</th><td valign="top" class="ep_row">12 Sep 2007</td></tr><tr><th valign="top" class="ep_row">Last Modified:</th><td valign="top" class="ep_row">08 Feb 2008 16:03</td></tr><tr><th valign="top" class="ep_row">ePrint Statistics:</th><td valign="top" class="ep_row"><a target="ePrintStats" href="/es/index.php?action=show_detail_eprint;id=1902;">View statistics for this ePrint</a></td></tr></table><p align="right">Repository Staff Only: <a href="http://eprints.utas.edu.au/cgi/users/home?screen=EPrint::View&eprintid=1902">item control page</a></p> </td></tr></table> </div> <!-- InstanceEndEditable --></td> </tr> <tr> <td><!-- #BeginLibraryItem "/Library/footer_eprints.lbi" --> <table width="795" border="0" align="left" cellpadding="0" class="footer"> <tr valign="top"> <td colspan="2"><div align="center"><a href="http://www.utas.edu.au">UTAS home</a> | <a href="http://www.utas.edu.au/library/">Library home</a> | <a href="/">ePrints home</a> | <a href="/contact.html">contact</a> | <a href="/information.html">about</a> | <a href="/view/">browse</a> | <a href="/perl/search/simple">search</a> | <a href="/perl/register">register</a> | <a href="/perl/users/home">user area</a> | <a href="/help/">help</a></div><br /></td> </tr> <tr><td colspan="2"><p><img src="/images/eprints/footerline.gif" width="100%" height="4" /></p></td></tr> <tr valign="top"> <td width="68%" class="footer">Authorised by the University Librarian<br /> © University of Tasmania ABN 30 764 374 782<br /> <a href="http://www.utas.edu.au/cricos/">CRICOS Provider Code 00586B</a> | <a href="http://www.utas.edu.au/copyright/copyright_disclaimers.html">Copyright & Disclaimers</a> | <a href="http://www.utas.edu.au/accessibility/index.html">Accessibility</a> | <a href="http://eprints.utas.edu.au/feedback/">Site Feedback</a> </td> <td width="32%"><div align="right"> <p align="right" class="NoPrint"><a href="http://www.utas.edu.au/"><img src="http://www.utas.edu.au/shared/logos/unioftasstrip.gif" alt="University of Tasmania Home Page" width="260" height="16" border="0" align="right" /></a></p> <p align="right" class="NoPrint"><a href="http://www.utas.edu.au/"><br /> </a></p> </div></td> </tr> <tr valign="top"> <td><p> </p></td> <td><div align="right"><span class="NoPrint"><a href="http://www.eprints.org/software/"><img src="/images/eprintslogo.gif" alt="ePrints logo" width="77" height="29" border="0" align="bottom" /></a></span></div></td> </tr> </table> <!-- #EndLibraryItem --> <div align="center"></div></td> </tr> </table> </body> </html>