Microbial biodiversity assessment of artificial microhabitatsexamples from Egyptian archaeological sites

  1. Samah Mohamed Rizk
Supervised by:
  1. Clara Urzi Director
  2. Rosa María Ros Espín Director
  3. Olaf Werner Director

Defence university: Universidad de Murcia

Year of defence: 2021

Committee:
  1. María Asunción Morte Gómez Chair
  2. Jairo Patiño Llorente Secretary
  3. Jesús Muñoz Fuente Committee member

Type: Thesis

Abstract

The general objective of this doctoral thesis was to know the microbial diversity present in microhabitats of archaeological sites with high cultural value, constantly exposed to harsh and arid environmental conditions. The pyramids of Djoser and Lahun were chosen to carry it out, two of the oldest and largest in the Memphis necropolis of ancient Egypt. The study focused on the bacteria and fungi that inhabit the stones and rocks of these archaeological sites, known for their acronyms in English, SIB (stone-inhabiting bacteria) and RIF (rock-inhabiting fungi). Microbial comparative studies were carried out using amplicon-based metabarcoding analysis, traditional in vitro culture isolation methods and techniques of epifluorescence microscopy. The metagenomic analysis of both pyramids allowed the identification of 644 species of bacteria and 204 of fungi. In isolation tests, 28 bacterial and 34 fungal species were identified. A total of 19 species of bacteria and 16 of fungi were exclusively culture-dependent, while 92 species of bacteria and 122 of fungi were culture-independent. The most abundant bacteria were of the Bacillus, Blastococcus and Planococcus genera. The most abundant fungi were Knufia karalitana and Pseudotaeniolina globosa. The morphological, physiological and molecular characterization of the strain of the black meristematic fungus P. globosa, isolated in the Djoser pyramid, was carried out and compared with an Italian strain. All data confirmed the conspecificity of the two isolates. However, the Egyptian one was able to grow under broader ranges of temperature and pH than the Italian, as well as extreme levels of salinity, and showed much more tolerance to heat and UV radiation. Based on the phylogenetic results using five markers (ITS, nrSSU/18S, nrLSU/28S, BT2 and RPB2), P. globosa was attributed to the family Teratosphaeriaceae (Capnodiales). The Egyptian strain can be considered a biovar well-adapted to extreme and harsh environments. In addition, two specific molecular markers were designed to detect inhabiting Actinobacteria (SIAb) the presence of stone quickly and inexpensively prior to further testing. The search for markers focused on the pathway for the synthesis of mycosporine-like amino acids (MAAs). Through “genomic mining”, two candidate genes were obtained: a homologue of a key gene in the MAAs and the shikimate pathways known as DAHP II (aroF) and another homologue of the Chorismate mutase gene (cm2). Both were found mainly in Actinobacteria. After calibration, the newly designed primers were successfully applied to environmental DNA extracted from the pyramids of Djoser and Lahun using quantitative PCR technique. It was concluded that the microbial diversity in both pyramids was higher than expected, considering the harsh conditions of the sampling sites. It was confirmed that the best methodological approach to study a complex microbial community is through the combination of microscopy and molecular identification for culture-dependent microbes and metagenomic methods for culture-independent ones. The potential bio-deteriorating effect of some of the SIBs and RIFs found on such an important cultural heritage sites requires attention to design conservation plans and find solutions to limit their presence. The development of new identification methods for SIAb allows a greater understanding of the diversity of organisms present in outdoor monuments, as it can lead to the discovery of new species or biovariants. The knowledge of the precise mechanisms of microorganisms to inhabit monuments and their role in biodeterioration is little studied, which requires more research in the future, both at the genomic, transcriptomic and metabolic levels using approaches based on next generation sequencing and bioinformatics.