# Microbial Activity in Subterranean Ecosystems: Recent Advances
## 3 The Search of Antibiotics Produced by Subsurface Bacteria
### Antibiotics from Subsurface Bacteria
Methodology based on heterologous expression and subsequent screening has been widely implemented in several species of the genus _Streptomyces_ and other actinobacteria for secondary metabolism expression and subsequent identification of bioactive compounds [93]. Culturing and isolation of microorganisms has been the primary methodology in new antibiotics discovery but, although nowadays it is extensively used, this approach is biased by the impossibility of extrapolating the subsurface microbiome to the laboratory. In fact, the majority of bacterial species cannot grow in laboratory conditions, an issue that could have led to disregarding a huge amount of new antibiotics synthetized by uncultured bacteria [87]. To amend the culture-dependent bias, metagenomic mining and metatranscriptomics should raise the chance to acquire novel bioactive compounds directly from the subterranean ecosystems. Metagenomics allows the study of genomes from non-culturable bacteria by means of sequencing the in-situ collected DNA and the subsequent bioinformatic analyses for assembly, binning, and annotation of the genomes of bacteria present in the sample. As a result of the treatment of metagenomic data, potential biosynthetic gene clusters are set in libraries and expressed by heterologous hosts to evaluate and validate their bioactivity. Metotranscriptomics have a double impact in microbiome studies--on one hand, RNA sequencing can focus on the metabolically active bacteria, discriminating the ancient DNA and latent or inactive bacteria that could add \"noise\" to the study, since the variation of relative abundance and presence of bacterial communities has been observed, even at the phylum level, in rRNA 16S studies when comparing total and metabolically active communities using both cloning and NGS analyses [33; 94]. Thus, efforts of bioactivity analyses could be more exhaustive and accurate. On the other hand, interactions inter- and intraspecies are the best frames to investigate the bioactivity of bacteria in competition with other species sharing the same niche. In the last decade, a few projects have been funded by the European Commission and National Organizations. They are SeaBioTech ([[https://spider.science.strath.ac.uk/seabiotech/index.php](https://spider.science.strath.ac.uk/seabiotech/index.php)]([https://spider.science.strath.ac.uk/seabiotech/index.php](https://spider.science.strath.ac.uk/seabiotech/index.php))), Marex ([[https://www.marex.fi/](https://www.marex.fi/)]([https://www.marex.fi/](https://www.marex.fi/))), PharmaSea ([[http://www.pharma-sea.eu/pharmasea/](http://www.pharma-sea.eu/pharmasea/)]([http://www.pharma-sea.eu/pharmasea/](http://www.pharma-sea.eu/pharmasea/))), FucoSan ([[https://www.fucosan.eu/en/](https://www.fucosan.eu/en/)]([https://www.fucosan.eu/en/](https://www.fucosan.eu/en/))), Tascmar ([[https://cords.europa.eu/project/id/634674](https://cords.europa.eu/project/id/634674)]([https://cords.europa.eu/project/id/634674](https://cords.europa.eu/project/id/634674))), and Probio ([[https://www.vbliz.be/en/news?p=show&id=8386](https://www.vbliz.be/en/news?p=show&id=8386)]([https://www.vbliz.be/en/news?p=show&id=8386](https://www.vbliz.be/en/news?p=show&id=8386))). All of them focused their activities on marine organisms (bacteria, algae, invertebrates, etc.). Activities and results can be found in their respective web pages. As far as we know no project on terrestrial microorganisms is ongoing, other than a recent research project launched with the aim of studying the biodiversity of extreme environments such as abandoned and active mines in Euroregion A3 (Alentejo, Algarve, and Andalucia) [95]. The mines (pyrite, manganese, copper, etc.) to be investigated are located in the Iberian Pyrite Belt. The Iberian Pyrite Belt is one of the World's largest accumulations of mine wastes and acidic mine waters from drainages, which have caused severe pollution by the low pH and presence of dissolved metals.