Spectrophotometric quantification of chlorosomal bacteriochlorophyll in intact cells of green sulphur bacteria: Monocultures and natural water.

In oceanographic and limnological studies, spectrophotometry combined with extraction of pigments by organic solvents became a routine procedure for chlorophyll (Chl) quantification in algae and cyanobacteria from oxic layers. However, for bacteriochlorophyll (BChl) of phototrophic bacteria inhabiting the oxic-anoxic interface and deeper anoxic zones, photometric determination of pigment becomes complicated because of overlapping absorption peaks from different chlorophylls, bacteriochlorophylls and phaeopigments in the solvent. The work is aimed to develop a new technique of BChl estimation in aquatic environments and to test this method using natural water samples with green sulphur bacteria (GSB, Chlorobiaceae). Absorption measurements were carried out using a laboratory spectrophotometer on monocultures of green-coloured as well as brown-coloured GSB in water (19 strains), along with natural water samples from the White Sea region containing either green-coloured or brown-coloured GSB. The far-red absorption peak with a maximum at 720-725 nm attributed to chlorosomal BChl in intact cells had practically the same position for all studied cultures. Its peak area was determined for each sample and compared with the total chlorosomal BChl content estimated from absorption spectra of extracts prepared from the same specimen. For all samples containing either green-coloured or brown-coloured GSB a good correlation between the peak area and BChl concentrations was found, with different regression slopes, however. Differences between the slopes were related to variations on mass-specific absorption coefficients between BChl. We provide formulas to evaluate the BChl content in intact GSB cells using absorption measurements performed without sample pre-treatment and pigment extraction. The suggested method can be used in communities where one type of microorganisms predominates (either green-coloured or brown-coloured GSB).在海洋学与湖沼学研究中，结合有机溶剂萃取色素的分光光度法，已成为定量测定含氧层中藻类与蓝细菌叶绿素（Chlorophyll, Chl）的常规手段。然而，对于栖息于含氧-缺氧界面及更深缺氧区域的光合细菌所产生的细菌叶绿素（Bacteriochlorophyll, BChl）而言，由于溶剂中不同叶绿素、细菌叶绿素与脱镁色素的吸收峰相互重叠，导致该色素的光度法测定变得复杂。本研究旨在开发一种可用于水生环境中细菌叶绿素定量的新技术，并采用携带绿硫细菌（Green Sulphur Bacteria, GSB，绿菌科（Chlorobiaceae））的天然水样对该方法进行验证。研究采用实验室分光光度计，对水体中19株绿色及棕色型绿硫细菌纯培养物开展吸光度测定，同时对取自白海区域、携带绿色或棕色型绿硫细菌的天然水样进行相同检测。所有受试培养物中，对应完整细胞内菌绿素体细菌叶绿素的远红外吸收峰峰值位置均集中于720~725 nm区间，且峰值位置几乎无差异。研究人员测定了每个样本的该吸收峰面积，并将其与从同一样本制备的萃取物吸光度光谱估算得到的总菌绿素体细菌叶绿素含量进行对比。针对所有携带绿色或棕色型绿硫细菌的样本，吸收峰面积与细菌叶绿素浓度之间均呈现良好的相关性，但二者的回归斜率存在差异。斜率差异与不同细菌叶绿素的质量比吸光系数变化相关。本研究提供了无需对样本进行预处理及色素萃取、仅通过吸光度测定即可估算完整绿硫细菌细胞内细菌叶绿素含量的计算公式。该方法可应用于以单一微生物类群（绿色或棕色型绿硫细菌）为主的群落样本检测。