討論

Winkler技術(shù)是估算浮游生物系統(tǒng)中細(xì)菌呼吸最常用的技術(shù)。該技術(shù)具有較高的靈敏度（見(jiàn)表2），但其缺點(diǎn)是無(wú)法隨時(shí)間連續(xù)監(jiān)測(cè)氧氣濃度。呼吸通常根據(jù)初始和最終氧氣濃度之間的差異計(jì)算，假設(shè)在孵化期間氧氣呈線(xiàn)性減少。先前的研究已經(jīng)表明，長(zhǎng)期培養(yǎng)過(guò)程中氧氣的減少并不總是線(xiàn)性的，但可以表現(xiàn)出不同的模式，如指數(shù)衰減或指數(shù)增加（Biddanda et al.1994；Pomeroy et al.1994）。此外，盡管靈敏度很高，但通常需要較長(zhǎng)的孵育時(shí)間來(lái)檢測(cè)顯著的呼吸速率，特別是在低營(yíng)養(yǎng)水域中，在那里孵育可長(zhǎng)達(dá)36小時(shí)。這些長(zhǎng)時(shí)間孵化的主要后果有充分的記錄；這包括細(xì)菌數(shù)量和活性的變化（見(jiàn)del Giorgio和Cole 1998年的綜述），以及群落組成的變化（Massana等人，2001年；Gattuso等人，2002年）。

表2. 測(cè)量浮游生物環(huán)境中氧濃度的不同方法

使用氧氣微探針測(cè)量細(xì)菌呼吸可以解決離散測(cè)量中遇到的主要問(wèn)題之一：在黑暗培養(yǎng)期間監(jiān)測(cè)氧氣減少。在這項(xiàng)研究中進(jìn)行的27項(xiàng)測(cè)量中，只有9項(xiàng)顯示出氧濃度的線(xiàn)性下降，其他的顯示出某種程度上與水的營(yíng)養(yǎng)狀態(tài)相關(guān)的趨勢(shì)。這種監(jiān)控有兩個(gè)主要優(yōu)點(diǎn)。首先，通過(guò)跟蹤氧濃度與時(shí)間的關(guān)系，可以檢測(cè)到顯著耗氧量的開(kāi)始。由于采用了保護(hù)陰極（Revsbech 1989），氧氣微探針不會(huì)消耗氧氣，并且顯示出約0.1μM O2的高精度，該值與在高精度Winkler測(cè)量中觀(guān)察到的值相似（見(jiàn)表2）。然而，這種高靈敏度被背景噪聲抵消，背景噪聲通常發(fā)生在用微探針測(cè)量氧氣的過(guò)程中。因此，在浮游水域進(jìn)行氧氣測(cè)量時(shí)，0.1μM的理論精度實(shí)際上降低到0.5μM O2。

第二個(gè)優(yōu)點(diǎn)是，一旦發(fā)現(xiàn)顯著的氧氣減少，就可以大大縮短培養(yǎng)時(shí)間，從而在記錄足夠的數(shù)據(jù)點(diǎn)時(shí)停止培養(yǎng)。因此，通過(guò)最小化瓶子效應(yīng)和伴隨的群落變化，在盡可能接近初始原位條件的條件下進(jìn)行測(cè)量。

然而，氧微探針的精度不足以測(cè)量培養(yǎng)時(shí)間短的貧營(yíng)養(yǎng)水體中的細(xì)菌呼吸。對(duì)貧營(yíng)養(yǎng)水體中氧濃度的監(jiān)測(cè)表明，只有在培養(yǎng)過(guò)程中細(xì)菌活性和生物量增加后，氧微探針才能測(cè)量到氧濃度的降低（圖4B）。這清楚地表明，這些水域的呼吸測(cè)量仍然存在問(wèn)題，因?yàn)槟壳斑€沒(méi)有靈敏度足以檢測(cè)這些非常低的原位呼吸率的技術(shù)。Gattuso等人（2002年）提出了替代技術(shù)的應(yīng)用，這將提供更高的氧敏感性，因此可能大大縮短培養(yǎng)時(shí)間，例如使用膜入口離子阱質(zhì)譜法（Cowie和Lloyd，1999年）來(lái)估計(jì)呼吸速率。

評(píng)論和建議

BGE的測(cè)定需要估計(jì)細(xì)菌產(chǎn)量。這通常是通過(guò)使用放射性標(biāo)記的亮氨酸或胸腺嘧啶核苷測(cè)量蛋白質(zhì)或DNA合成速率來(lái)完成的，盡管也可以使用細(xì)菌豐度和大小的變化。通過(guò)加入放射性示蹤劑來(lái)估計(jì)細(xì)菌產(chǎn)量可以在很短的培養(yǎng)時(shí)間內(nèi)完成，并且被認(rèn)為是原位率的一個(gè)很好的代表。然而，BGE是根據(jù)比用于測(cè)定細(xì)菌產(chǎn)量的時(shí)間更長(zhǎng)的培養(yǎng)時(shí)間內(nèi)估計(jì)的細(xì)菌呼吸來(lái)計(jì)算的。因此，BGE是根據(jù)在兩種不同培養(yǎng)條件下估計(jì)的兩種代謝過(guò)程的速率來(lái)計(jì)算的，這可能會(huì)使其產(chǎn)生偏差（即，在短時(shí)間間隔內(nèi)測(cè)量的生產(chǎn)速率可能與更長(zhǎng)時(shí)間范圍內(nèi)的呼吸速率不一致）。根據(jù)培養(yǎng)期間細(xì)菌豐度的變化估算細(xì)菌凈產(chǎn)量，以進(jìn)行呼吸測(cè)量，這是一種替代解決方案。通過(guò)使用非破壞性方法測(cè)量氧氣變化，可以在培養(yǎng)結(jié)束時(shí)獲得子樣本，以確定細(xì)菌的凈生物量。這樣，兩個(gè)過(guò)程將以相同的時(shí)間尺度和相同的孵化條件進(jìn)行估計(jì)。

通過(guò)連續(xù)監(jiān)測(cè)細(xì)菌呼吸測(cè)量期間的氧氣變化來(lái)縮短培養(yǎng)時(shí)間的可能性需要以足夠的精度確定細(xì)菌凈生物量的產(chǎn)生。為了達(dá)到所需的靈敏度，使用表觀(guān)熒光顯微鏡測(cè)定細(xì)菌數(shù)量需要對(duì)大量細(xì)菌進(jìn)行計(jì)數(shù)，并使用多個(gè)復(fù)制品，特別是在貧營(yíng)養(yǎng)水域。這將大大增加與測(cè)量相關(guān)的工作量。流式細(xì)胞術(shù)可能是測(cè)定呼吸培養(yǎng)期間細(xì)菌凈生物量的一種替代技術(shù)。與表面熒光顯微鏡相比，該技術(shù)提供了一種更高靈敏度的細(xì)菌數(shù)量測(cè)量方法（Troussellier等人，1999年；Lemarchand等人，2001年）。此外，流式細(xì)胞術(shù)可用于在培養(yǎng)開(kāi)始和結(jié)束時(shí)估計(jì)細(xì)胞的生物體積，甚至蛋白質(zhì)含量（Zubkov等人，1999年），從而更好地計(jì)算細(xì)菌凈產(chǎn)量，因?yàn)樵贐GE測(cè)定的培養(yǎng)過(guò)程中，經(jīng)常報(bào)告細(xì)菌細(xì)胞生物體積的變化（Gattuso等人，2002年）。

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