We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

ガラス製品とフィルターハウジングの調製<ol style="margin-left: 40px;"><li>特に断りのない限り、15分間121℃で、すべてのガラス製品、フィルターハウジングとソリューションを滅菌する。無菌性を確保するために、滅菌前にアルミ箔またはテープで保護された紙のいずれかですべての開口部または露出した表面を覆う。 </li><li> 1Lのサイドアームの三角フラスコに、フィルタハウジング（直径47mm）を取り付けることにより、ろ過装置を組み立てる。 47ミリメートル直径電気陰性/電気陽性ディスクフィルター：必要なフィルターを収集します。 </li><li> （凝集pHは3.5で集計しない;フロック溶液のpHを3.5に低下させ、粒子、または非凝集生成）1.5％牛肉エキス1リットル調製牛肉抽出物15gを溶解させて、0.05 Mグリシンを脱イオン水1 L及び3.75グラムのグリシンを添加する。 注：非凝集牛肉抽出物は、前三次濃度にセライトの追加が必要になります。 </li><li>溶出添加剤のナトリウムpolyphを追加0.1％の濃度で、牛肉エキスにosphate。 </li><li>牛肉エキス、所望のpHへのpHの溶液を混合し、滴下し、1 M塩酸溶液を加える。 121℃で15〜30分間のオートクレーブ牛肉エキス。 </li><li>細かい、中、または（非凝集牛肉エキスのみで使用するための）大粒子セライトの0.1グラムを測定します。 </li><li>エルレンサイドアームフラスコに、真空排気口に互換性のあるチューブを取り付けることにより、真空/吸引を準備します。 </li></ol>ソリューションおよびウイルスストックの調製<ol style="margin-left: 40px;"><li> 1MのHClの1 Lを準備します。 </li><li> 1 M NaOHを1リットルを準備します。 </li><li> 1×PBS溶液を調製する（137ミリモルのNaCl、2.7のKCl、1.47 mMのK 2 PO 4、及び4.3ミリモルのNaH 2 PO 4）。 <ol><li> 1 M NaOHを用いて9.0に1×PBSのpHを調整する。 </li></ol></li><li>準備し、9のPBS（pHのストックウイルスの1ミリリットルを混合することにより、10 4〜10 5最確数（MPN）ミリリットル-1にストックウイルスを希釈7）、以前に8,13に記載のように。 1ミリリットルのアリコートで-80℃で保存。 <ol><li>潜在的な汚染物質を除去するために、シリンジフィルター（0.22μmの孔径）によってウイルスストックを滅菌する。 </li></ol></li><li>脱塩素水道水<ol><li>メジャー滅菌を使用して滅菌水道水1Lメスシリンダーおよび磁気撹拌棒を含む2リットルのビーカーに注ぐ。 </li><li>チオ硫酸ナトリウムの0.7グラムを加え、顆粒が溶解するまで混合する。必要であれば、1 M HClまたは1MのNaOH溶液を用いて7-7.5に水道水のpHを調整する。 </li><li>融解ウイルススパイク​​（1ml）および脱塩素滅菌水道水1Lにスパイクの全体積を加える。 10分間混ぜる。 </li><li>スパイクされたサンプルと同じ方法で、各実験とプロセスとメソッドの空白（アデノウイルスのない脱塩素水道水）を準備します。 </li></ol></li></ol>ミリポアフィルタ装置の調製<ol style="margin-left: 40px;"><li>フィルターハウジングの上に無菌のカバーを外します。確保濾過中のサンプルの漏れを回避するために、フィルタハウジング上部ボウルと下部フィルタスクリーンとの間の適切なフィット。 </li><li> 1Lの三角フラスコから無菌のカバーを外します。フィルタユニットのコルクは、良好なシールを作成、三角フラスコの上部開口にぴったりとフィットしていることを確認してください。 </li><li>下部フィルタ画面からフィルターユニットの上部ボウルを外し、滅菌ピンセットで画面上で真正面から47ミリメートル電気陽性フィルタディスクを置く。下の画面にフィルターハウジングボウルを取り付け、反時計回りにねじることによって所定の位置にロック。 </li></ol> 4.水道水濾過（一次濃縮） <ol style="margin-left: 40px;"><li>ウイルスの100ミリリットルを測定し、滅菌メスシリンダーを使って水道水をスパイクした。 </li><li>ウイルスの100mlを47ミリメートルプリーツガラス/セルロースフィルタ又はナノアルミナ/グラスファイバーディスクフィルターのいずれかを含むフィルタハウジング内に水道水を混ぜ入れる。 </li><li>水がゆっくりとフィルターを通過することを許可する。 O残りの残差を除去するために穏やかな真空を適用しますフィルター表面からfの水道水。 </li><li>トリプシン処理を使用する場合は、表面をフィルタリングし、5分間インキュベートし、0.25％トリプシンの5ミリリットルを追加します。 </li><li>滅菌脱イオン水50mlを用いてフィルタ表面からトリプシンを洗う。 </li></ol>フィルターから5.ウイルス溶出<ol style="margin-left: 40px;"><li> 2 L三角フラスコからフィルターハウジングを外し、250ミリリットルサイドアームフラスコに配置します。 </li><li>メスシリンダーを使用して、牛肉エキスの100ミリリットルを測定します。ゆっくりとフィルターハウジングに牛肉エキスの100ミリリットルを注ぐ。 </li><li>小さな（250ミリリットル）の三角フラスコに牛肉エキスをキャプチャ、牛肉エキスがフィルターを注ぐようにします。フィルタからの残留牛肉エキスを通して引くために真空を適用します。 </li></ol> 6.セライト二次濃縮<ol style="margin-left: 40px;"><li>撹拌プレートに溶出したウイルスを含むトランスファーフラスコ。わずかな渦を作成するために混合、撹拌プレート上の滅菌磁気撹拌棒及び場所を追加する。 </li><li>セライトの0.1グラムを追加します。牛肉エキスの100ミリリットルに粉末、セライトを分散させることができます。牛肉エキスに無菌のpHプローブを配置します。 </li><li> pHが4.0を達成するために牛肉エキスに賢明1MのHClをドロップ追加します。 10分間かけて徐々に混在させることができます。 </li><li> 2Lの三角フラスコを用いて、（上述したように）フィルタハウジング上に47ミリメートルプレフィルタを配置する。 </li><li>プレフィルター上で100ミリリットル牛肉エキス/セライト混合物を注ぐ。牛肉エキスが通過注ぐできるようにします。フィルターから残留牛肉エキスを通じて引っ張ってわずかな真空を適用します。 </li><li>フィルターハウジング注ぎ口の端に金属チューブクリップを配置します。金属管クリップに滅菌15ミリリットルポリプロピレンコレクションチューブを取り付けます。バック三角フラスコにフィルターハウジングを置きます。 </li><li>プレフィルター上に回収し、セライト上で1×PBS緩衝液5ml、pHが9.0を加える。 </li><li> PBSは、プレフィルターを介してドリップすることを許可する。 15ミリリットルのコレクションチューブに残留PBSを引っ張ってわずかな真空を適用します。 </li></ol> 7.オーガニック凝集二次濃縮<ol style="margin-left: 40px;"><li>撹拌プレートに溶出されたウイルスを含有するビーカーに移す。牛肉エキス100mlに無菌の磁気撹拌棒を追加して、わずかに渦を作成するために混ぜる。 </li><li>牛肉エキスに無菌のpHプローブを配置します。 </li><li> 1 M HClを牛肉エキスに滴下し、pHを3.5に調整追加します。 30分間混ぜる。 </li><li> 4ºCで15分間2500×gで250ミリリットル遠心コニカルチューブと遠心分離機に溶出液を注ぐ。 </li><li>上清をペレットを破壊しないようにしないように注意しながら捨てる。 1×PBS、pHが9 5mlにペレットを再懸濁する。 </li><li> 4ºCで10分間4000×gでサスペンションを遠心。上清を捨て、ペレットを捨てる。 </li><li> 7-7.5のpHを調整し、フィルターを-80℃で0.22μmのシリンジフィルターを通して滅菌し、凍結。 </li></ol> 8.ウイルス核酸抽出<ol style="margin-left: 40px;"><li>製造業者の説明書に従って、適切な市販のキットを用いてウイルス核酸を抽出する。 </li></ol> &lt;Pクラス= &quot;jove_title&quot;&gt; 9。のAdV 40/41定量PCR <ol style="margin-left: 40px;"><li> 8,13が公開されているプライマーおよびプローブ、ならびに反応混合物及び熱サイクルプロファイルを使用。 注：分析のための検出の推定限界は反応あたり約5 qPCRの単位である。 </li><li>以下のような濃度でPCR増幅混合物の成分を追加します。10 mMトリス（pHは8.3）、50mMのKCl、4.5のMgCl 2、10mMのdNTPを、10μMのプライマーおよび1μMのプローブおよびポリメラーゼ0.5μlの45の最終的なボリュームになるようμlを96ウェルPCR反応プレート内のウェルの適切な数に加えることができる。 </li><li> 96ウェル、速い反応PCRプレートの各ウェルに、適切なPCR増幅混合物の45μlの負荷。 </li><li> DNAの5μl加えPCRプレートの適切なウェルにサンプルを抽出した。 </li><li>耐熱カバーシールでプレートをカバーし、PCRプレートのウェルの底に任意の液滴を移動させるために、プレートをスピン。 </li><li> follとしてPCR増幅サイクルを設定するOWS：10分間95℃、1分15秒、60℃、95℃の40サイクル。 </li></ol> 10.データ分析<ol style="margin-left: 40px;"><li> 5：1：1に及ぶサンプルの連続5倍希釈の3回の測定を分析625希釈範囲のウイルス粒子の最確数（MPN）を決定するために、以前に8,13に記載のように。 </li><li> 5：1：1に及ぶウイルス実験スパイクの連続5倍希釈を分析15625希釈範囲のウイルス粒子の最確数（MPN）を決定するために。 </li><li> EPA MPN計算器（http://www.epa.gov/nerlcwww/online.html）を使用して、希釈液のタイプ（1：5または1:10）を説明するために計算機を調整し、実行さ希釈液の数、および数希釈系列でサンプルを複製する。 </li><li> などボリューム（ 例えば 、1ミリリットル、100ミリリットル、の単位に正規化することにより、その後、データの10変換をログ実行</EM&gt;）。試料中に存在するウイルス粒子の数を割った実験の回収率を計算し、実験スパイク中に存在するウイルス粒子の数に集中する。 </li><li>異なる実験変数の影響を評価する（ 例えば、別のセライト種類、pH範囲、別のフィルタタイプと異なる二次濃縮技術）（ 例えば 、t検定、1つまたは2つのパラメトリックまたはノンパラメトリック統計検定を行うことでのAdV回収にデータの分布と実験的な設計に依存）ANOVAを分割バグレー。 </li></ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Round+robin+investigation+of+methods+for+the+recovery+of+poliovirus+from+drinking+water.">Round robin investigation of methods for the recovery of poliovirus from drinking water.</a> Applied and Environmental Microbiology. 47, 144-150 (1984).</li><li>Schwab, K. J., De Leon, R., Sobsey, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+purification+of+beef+extract+mock+eluates+from+water+samples+for+the+detection+of+enteroviruses,+hepatitis+A+virus,+and+Norwalk+virus+by+reverse+transcription-PCR.">Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses, hepatitis A virus, and Norwalk virus by reverse transcription-PCR.</a> Applied and Environmental Microbiology. 61, 531-537 (1995).</li></ol>

No competing financial interests. 
The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

陽性のフィルタは、水からのウイルスを濃縮するのに有用である。しかし、これらのフィルタは、順番にそれらの有効性を変えることができ、その構造と組成が異なることができる。この問題を配合、カプシド構造と料金は、最適な回復15を確保するように調整することが濃縮技術を必要とするウイルス株の間で変動する。既存の濃縮技術（ 例えば、電気陽性フィルタ、牛肉?...

人間の腸内ウイルスは、水系感染症1-3の重要な原因物質であるが、濃度のないそれらの検出を困難にし、一般的に汚染された環境水中の低い数で存在する。ウイルスを濃縮するために使用される手順は、典型的には濾過フィルター溶出したステップ、およびフィルタ溶出液の二次濃縮を含む。一般的な濾過手順は、そのような電気陽性フィルター（最近4,5にレビュー）のような荷電膜の使用に依存している。これらのフィルタは、フィルタ表面との間の静電相互作用（正に荷電）、および標的ウイルス粒子（負に帯電した）を使用して水に懸濁させたウイルスを捕捉するに依存する。市販されている二つの陽性のフィルタは、この技術は、ガラス/セルロース、ナノアルミナ/ガラス繊維フィルターに依存している。ガラス/セルロースフィルターのコストは、ガラス/ CEの使用を制限するナノアルミナ/ガラス繊維の最大10倍であるルーチンウイルス監視のためのフィルタをllulose。最近の研究では、違いが安く、フィルタの代替の使用を正当化する、周囲の水の6,7からのエンテロウイルスの回復にこれら二つのフィルタの間に、公称である結論に達した。そのような電気陰性及びグラスウールフィルタのような他のフィルタオプションが、それらはいずれかの源水（電気陰性フィルタ）の前処理を必要とするか、または（グラスウールフィルタ）市販されていない、研究されている。ウイルス濃縮手順の開発は、主に水からのウイルス回収率を向上させるために一次濃縮技術（フィルタ）を最適化することに焦点を当ててきた。しかし、ボリュームミリリットルため、典型的には1 Lから溶離剤の量を減少させる二次濃縮手順は、また、ウイルスの回収8に重大な影響を与えることができる。 腸内ウイルスの二次濃度は、典型的には、牛肉エキス、いくつかのタイプとして凝集剤（有機floccuに依存しています設置と）またはスキムミルク凝集9-12フィルタ表面からウイルス粒子を除去する。最近、セライト（微粒子）を添加して結合された牛肉エキスを使用して別の二次濃縮手順は、アデノウイルス、エンテロウイルス、およびノロウイルス8,13,14を回収するための有望性を示している。セライト濃度に付着し、懸濁液のpHを変化させることによって、粒子（フロックまたはセライト）から放出されるウイルス粒子中の有機凝集法と同様の原理で動作します。これら二つの二次濃縮技術との比較は、スパイクアデノウイルス（のAdV）タイプ40及び41 8の回復に評価されてきた。本研究では、2つの二次濃縮技術は、アデノウイルスの回復の統計学的に類似していたと結論付けた。しかし、有機凝集法は、30分を要する。 pH3.5でのインキュベーション、セライト技術はpH4.0で短いインキュベーション（10分）を必要とする。有機flocculatイオンはまた、コントラストセライト技術が懸濁液からセライト粒子を分離するために、基本的な実験装置（減圧濾過）を使用して、第三級濃縮中フロック粒子を収集するために、高価な実験装置（遠心分離機）の使用を必要とする。 フィルタおよび二溶出技術の特定の組み合わせはまた、ウイルス回収率に影響を与えることができる。ある研究では、プライマリ（電気陽性フィルタ）の特定の組み合わせと二次濃縮技術（セライトまたは有機凝集）はアデノウイルス13の回復の重要な影響を与えたと結論付けた。これらの知見は、最適化は、これらの技術を使用する場合に最適に与えられた水マトリックスから標的ウイルスを回収するために必要とされることを示唆している。最適化は、非常に多くの変数が（フィルタータイプ/ブランド、pH溶出液、セライト/有機凝集）が評価されるため、多くの研究者が積極的に避ける時間のかかる、骨の折れるプロセスです。 これはsのtudy、手順は、スパイク、ヒトアデノウイルスを用いて水からのウイルス濃度の最適条件を同定するために開発されたおそらく40,41菌株、各ウイルスタイプはユニークなカプシド形態および特定のキャプシド電荷を表示するので、濃度プロトコルは、すべてのウイルスのために最適化する必要があるかもしれない最適なウイルスの回復を達成するために、標的とする。 ）二次濃縮、および3としての評価1）2、続いて陽性フィルターディスクを用いて水道水にウイルス回収率を評価する）、セライト技術に対する確立された有機凝集法の評価：この研究は、バイのAdV 40,41濃度ためのアプローチを提供三次濃度の溶出バッファー。

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
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			<td height="21" style="height:21px;width:231px;">Adenovirus 40 stock</td>
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			<td height="21" style="height:21px;width:231px;">NanoCeram 47mm</td>
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			<td height="21" style="height:21px;width:231px;">1MDS 47mm</td>
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</table>

a small volume procedure for viral concentration from water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

<html>セライトの選択セライトの3つの異なるタイプの前最高のパフォーマンス変異体の選択に試験した。中サイズの粒子に罰金セライトは、最高のアデノウイルスの回収を生産した。大きなセライトの使用はAdV40 41（範囲32％-100％）（ 図1）の両方の回収率の低下をもたらした。アデノウイルス40の平均回収率は115％±28％の（中）と82％±53％で（大）粒子セラ?...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

水からのウイルス濃度のため、小容量の手順

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

a-small-volume-procedure-for-viral-concentration-from-water

Research

JoVE Journal

Environment

10.0K ビュー.  U.S. EPA. An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation. 

ビデオ: A Small Volume Procedure for Viral Concentration from Water

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

内部リン負荷の尺度として湖堆積物の研究室に決定されたリンフラックス

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

環境学

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

水 - パルス振幅変調（WATER-PAM）蛍光測定を用いて細菌/植物プランクトンの共培養を評価するために小音量バイオアッセイ

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

CO<sub&gt; 2</subテスト用のCO&gt;濃度勾配施設<sub&gt; 2</sub&gt;濃縮および草地生態系の機能に及ぼす土壌の影響

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

小さな汚染物質の免疫学的分析のための水試料の自動固相抽出ための簡単​​な方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to climate change and crop management. Quantitative size information on roots in their native environment is invaluable for studying root growth and environmental processes involving plants. X-ray computed tomography (XCT) has been demonstrated to be an effective tool for in situ root scanning and analysis. We aimed to develop a costless and efficient tool that approximates the surface and volume of the root regardless of its shape from three-dimensional (3D) tomography data. The root structure of a Prairie dropseed (Sporobolus heterolepis) specimen was imaged using XCT. The root was reconstructed, and the primary root structure was extracted from the data using a combination of licensed and open-source software. An isosurface polygonal mesh was then created for ease of analysis. We have developed the standalone application imeshJ, generated in MATLAB1, to calculate root volume and surface area from the mesh. The outputs of imeshJ are surface area (in mm2) and the volume (in mm3). The process, utilizing a unique combination of tools from imaging to quantitative root analysis, is described. A combination of XCT and open-source software proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information from the 3D data. This methodology of processing 3D data should be applicable to other material/sample systems where there is connectivity between components of similar X-ray attenuation and difficulties arise with segmentation.

A methodology for obtaining visual and quantitative root structure information from X-ray computed tomography data acquired in-soil is presented.

三次元根系のための抽出メトリック：イン土壌のX線コンピュータ断層撮影データからボリュームおよび表面分析

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

結合水文学、地球化学、および微生物学的調査のための土壌ライシメータ発掘

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

プラスチックペレットからの有機塩素系農薬の抽出と塑性解析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

排水のイベント中に水の流れ加重と農業用水路から浮遊微粒子をキャプチャ

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

単純な頻繁に浸水が発生した再構築ツリー手法を植栽

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

最適化された小型リン定量法を用いた粒状水酸化第二鉄に酸の吸着を確認する手順

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...