Identification of bacterial taxa in archaeological waterlogged wood

Franco Palla, Giovanna Barresi, Enza Di Carlo

Abstract


Summary
The microscopic and molecular techniques described in this study are aimed at understanding the degradation processes of the anatomical structure of submerged archaeological wood, correlating it to the degradation induced by bacteria. The SEM micrographs showed alterations of the wooden structure due to bacterial colonization, as well as the presence of pyrite framboids. The difficulty of extracting bacterial DNA from wooden fragments belonging to submerged finds is well-known, due to the presence of many inhibitors; this study describes some extraction and in vitro amplification protocols for wooden submerged finds. The results of the molecular investigations, based on the analysis of specific sequences of microbial genomic DNA enabled us to detect the presence of cellulolytic and ligninolytic bacteria, in addition to iron-oxidizing or sulfatereducing bacteria, otherwise undetectable by traditional in vitro culture methods.

Riassunto
Le tecniche microscopiche e molecolari impiegate nel presente studio sono finalizzate alla comprensione dei processi degradativi della struttura anatomica del legno archeologico sommerso correlandolo al degrado indotto da batteri. Le micrografie SEM evidenziano alterazioni della struttura lignea riconducibili a colonizzazione batterica, oltre la presenza di framboidi di pirite. La difficoltà di estrarre il DNA batterico dai frammenti lignei appartenenti a reperti sommersi è nota, a causa della presenza di molti inibitori; in questo studio sono riportati alcuni protocolli applicativi per manufatti lignei sommersi. I risultati delle indagini molecolari, basate sull’analisi di specifiche sequenze del DNA genomico microbico hanno permesso di rilevare la presenza di batteri cellulosolitici e ligninolitici, oltre a batteri ferro-ossidanti e solfato-riduttori, altrimenti
non rilevabili con le tradizionali metodiche colturali.

Résumé
Le but des techniques microscopiques et moléculaires utilisées dans cette étude est la compréhension des processus de dégradation de la structure anatomique du bois archéologique submergé, en lien avec la dégradation provoquée par les bactéries.
Les micrographies à balayage électronique font ressortir que les colonisations bactériennes provoquent des altérations de la structure du bois. On constate aussi la présence de framboïdes de pyrite. Il est bien connu qu’il est difficile d’extraire l’ADN bactérien des fragments de bois de vestiges archéologiques retrouvés sous l’eau, à cause de la présence de nombreux inhibiteurs ; cette étude ne mentionne que quelques-uns des protocoles appliqués aux pièces submergées en bois. Les résultats des études moléculaires fondées sur l’analyse de séquences spécifiques de l’ADN des gènes des bactéries ont permis de détecter la présence de bactéries cellulolytiques et ligninolytiques, mais aussi celle de bactéries ferro-oxydantes et sulfato-réductrices, dont la détection aurait été impossible avec les méthodes de culture traditionnelles.

Zusammenfassung
Die in dieser Studie angewandten mikroskopischen und molekularen Methoden zielen auf das Verständnis der Prozesse ab, die der Schädigung der anatomischen Struktur archäologischen, unter Wasser befindlichen Holzes zugrunde liegen, sowie deren Beziehung mit der Schadwirkung von Bakterien. Die SEM-Mikrographien zeigen Veränderungen der Holzstruktur, die auf die Kolonisierung mit Bakterien zurückzuführen sind, sowie das Vorhandensein von Pyrit-Framboiden. Die Schwierigkeit, aus Holzfragmenten von unter Wasser gelegenen Fundstücken die Bakterien-DNA zu extrahieren, ist bekannt, da zahlreiche Inhibitoren vorhanden sind. In dieser Studie sind einige Anwendungsprotokolle für unter Wasser liegende Manufakte abgedruckt. Die Ergebnisse der Molekularuntersuchungen durch Analyse von spezifischen Sequenzen des DNA-Genoms der Mikroben haben es gestattet, das Vorhandensein von Cellulose und Lignin abbauenden Bakterien sowie von eisenoxidierenden und Sulfat reduzierenden Bakterien nachzuweisen, die mit den herkömmlichen Kulturmethoden nicht erkannt werden können.

Resumen
Las técnicas microscópicas y moleculares empleadas en el presente estudio tienen como objetivo conocer los procesos de degradación de la estructura anatómica de la madera arqueológica sumergida comparándolos con el deterioro provocado por bacterias. Las micrografías SEM muestran alteraciones en la estructura lignaria atribuibles a colonización bacteriana, además de presentar framboides de pirita. Como es sabido, la presencia de muchos inhibidores dificulta la extracción del ADN bacteriano de los fragmentos lignarios pertenecientes a restos sumergidos. En este estudio se enuncian varios protocolos aplicables a materiales lignarios sumergidos. Los resultados de las investigaciones moleculares, fruto del análisis de secuencias específicas
de ADN genómico microbiano, han permitido identificar la presencia de bacterias celulolíticas y ligninolíticas, además de bacterias ferroxidantes y reductoras de sulfato, que no se hubieran podido detectar con los métodos de cultivo tradicionales.

简介
在这份研究中,显微镜技术和分子技术用于帮助理解浸水的考古出土木材,其解剖
学结构的降解过程,并将由细菌引起的降解联系其中。SEM显微镜除显示黄铁矿微粒
球的存在外,还会显示由于细菌增殖的造成的木质结构的改变。由于抑制剂的存在,
从水浸木材中提取细菌DNA的难度是众所周知的;在这份研究中引述了一些关于浸水
木制品的应用协议。基于微生物基因组DNA的特定序列,分子研究的结论显示其中不
仅有铁氧化菌和硫酸盐还原菌的存在,还有纤维分解菌和木质素分解菌的存在,这在
传统的培养法中是没有被发现的。

Резюме
Микроскопические и молекулярные методы, примененные в настоящем иссле-довании, нацелены на объяснение процессов разложения анатомического стро-ения археологической древесины, сопоставляя их с разложением, вызванным бактериями. На СЭМ-микрографиях показаны изменения структуры древесины, вызванные бактериальной колонизацией, а также наличие фрамбоидального пирита. Известны трудности, связанные с извлечением бактериального ДНК из фрагментов древесины, полученных из подводных объектов по причине присут-ствия многочисленных ингибиторов. В данном исследовании указаны несколь-ко протоколов, применимых к подводным деревянным изделиям. Результаты молекулярных исследований, основанных на анализе специфических после-довательностей геномной ДНК микробов, позволили выявить наличие целлю-лозолитических и лигнинолитических бактерий, а также железоокисляющих и сульфатредуцирующих бактерий, которые нельзя обнаружить при помощи традиционных методов разведения.


Keywords


submerged archeological wood; biodeterioration; SEM; molecular techniques

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DOI: 10.6092/issn.1973-9494/4748

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Copyright (c) 2014 Franco Palla, Giovanna Barresi, Enza Di Carlo

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