A Highly Diverse, Desert-Like Microbial Biocenosis on Solar Panels in a Mediterranean City

bioRxiv preprint doi: https://doi.org/10.1101/029660; this version posted November 30, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A highly diverse, desert-like microbial biocenosis on solar panels in a Mediterranean city Pedro Dorado-Morales1,2, Cristina Vilanova1, Juli Pereto´ 1,3, Francisco M. Codoner˜ 4, Daniel Ramon´ 2 and Manuel Porcar1, 5,* 1Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de Valencia,` Paterna 46980, Spain 2Biopolis S.L., Parc Cient´ıfic Universitat de Valencia,` Paterna, Valencia, Spain 3Departament de Bioqu´ımica i Biologia Molecular, Universitat de Valencia,` Burjassot 46100, Spain 4Lifesequencing S.L., Parc Cient´ıfic Universitat de Valencia,` Paterna, Spain. 5Fundacio´ General de la Universitat de Valencia,` Spain. *Address correspondence to Manuel Porcar: [email protected] P.D. and C.V. contributed equally to this work. 1 icroorganisms colonize a wide range Introduction 27 2 of natural and artificial environments 3 although there are hardly any data M Today, photovoltaic panels cover around 4000 square 28 4 on the microbial ecology of one the most kilometers, and are forecasted to be the world's main 29 5 widespread man-made extreme structures: electricity source by 2050 (http://www.epia.org). So- 30 6 solar panels. Here we show that solar pan- lar panels are unique biotopes characterized by a 31 7 els in a Mediterranean city (Valencia, Spain) smooth flat glass or glass-like surface, minimum wa- 32 8 harbor a highly diverse microbial commu- ter retention capacity and maximum sunlight expo- 33 9 nity with more than 500 different species per sure, all of which determine circadian and annual 34 10 panel, most of which belong to drought-, heat- peaks of irradiation, desiccation and heat. Extreme 35 11 and radiation-adapted bacterial genera, and natural habitats such as thermal vents, mountain 36 12 sun-irradiation adapted epiphytic fungi. The plateaus or hyper arid deserts are known to host 37 13 taxonomic and functional profiles of this mi- microbial biocenoses adapted to those particular se- 38 14 crobial community and the characterization lection pressures (1, 2, 3); and artificial or humanized 39 15 of selected culturable bacteria reveal the ex- environments, such as industrial reactors (4), radioac- 40 16 istence of a diverse mesophilic microbial com- tive waste (5) or oil spills (6) are also colonizable 41 17 munity on the panels surface. This bioceno- by specialized microorganisms. However, despite the 42 18 sis proved to be more similar to the ones in- popularity and the quick spreading of photovoltaic 43 19 habiting deserts than to any human or ur- panels, the microbial communities potentially asso- 44 20 ban microbial ecosystem. This unique mi- ciated to these human-manufactured devices have 45 21 crobial community shows different day/night not been described to date. Our report documents 46 22 proteomic profiles; it is dominated by reddish a complete bioprospection and characterization of 47 23 pigment- and sphingolipid-producers, and is the microbial community on photovoltaic panels of a 48 24 adapted to withstand circadian cycles of high Mediterranean city, using high throughput 16S/18S 49 25 temperatures, desiccation and solar radia- analysis, metagenomic sequencing, metaproteomics, 50 26 tion. and culture-based characterization of selected iso- 51 lates. 52 Page 1 of 13 bioRxiv preprint doi: https://doi.org/10.1101/029660; this version posted November 30, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Materials and Methods Strains stress tests 50 Each strain was subjected to a range of stress assays 51 2 Sampling to test tolerance to salinity, heat, low pH, and UV 52 3 Sampling was performed during the summer solstice radiation. Overnight liquid cultures were adjusted 53 4 of 2013 and 2014. Sampling consisted of a simple har- to an OD600 value of 0.03. Then, stress tests were 54 5 vesting procedure, by pouring sterile PBS (sodium carried out by plating several 20 L droplets of the 55 6 phosphate buffer) on the panel and immediately har- diluted culture on LB or marine agar with the fol- 56 7 vesting the liquid by strongly scraping the surface lowing modifications. In the case of salinity stress, 57 8 with a modified window cleaner with an autoclaved increasing amounts of NaCl (from 1 to 9% w/v) were 58 9 silicone tube measuring 5 mm in diameter. The re- added to the media (final concentration ranging from 59 10 sulting suspension was collected by using a sterile 1 to 26% w/v). To test pH resistance, culture media 60 11 plastic pipette and transferred to sterile Falcon tubes, was adjusted to pH 5, 6, and 8. In the case of heat, 61 12 placed on ice and immediately transported to the lab. plates were incubated overnight at 60 C; whereas 62 13 In 2013, nine samples from solar panels on the three resistance to radiation was tested by applying UV 63 14 campuses of the University of Valencia (Valencia, pulses of different length (30 s, 2 min, and 8 min) 64 - 15 Spain) were collected at noon (2 PM) and pooled. with a VL-4C lamp (254 nm, 340 W cm 2; Labolan, 65 16 Air temperature was 33 C and relative humidity was S.L., Spain). The XL1-Blue E. coli strain was used as 66 17 60%. In 2014, samples from three independent solar control. For each experiment, two independent repli- 67 18 panels in a single location (Faculty of Economics, cates were performed. In order to detect inhibition or 68 19 University of Valencia, Valencia, Spain) were har- synergistic effects between strains, experiments were 69 20 vested at noon (2 PM) and at night (4 AM) for the performed as described above and strain suspensions 70 21 proteomic studies, whereas three additional samples (20 ul) were closely (3 mm) placed on the same dish. 71 22 (panels 1, 2 and 3) were sampled and used for both 23 16S/18S rRNA taxonomic identification and metage- DNA isolation 72 24 nomics (Figure 1A). Air temperature and relative Selected DNA purification methods were used to pro- 73 25 humidity were 32 C and 56% (2 PM), and 23 C and cess the solar panels samples and DNA yields were 74 26 83% (4 AM). The average temperature of the panels compared (data not shown). Metagenomic DNA 75 27 surface during the sampling process (at 2 PM) was was isolated using the Power Soil DNA Isolation 76 28 51 C. The average solar irradiance in Valencia at 2 2 kit (MO BIO Laboratories) following the manufac- 77 29 PM is 461.3 W/m , whereas the accumulated solar 2 turers instructions with an additional pretreatment 78 30 irradiance during an average day is 19.6 kJ/m . with DNA-free lysozyme at 37 C for 10 min. The 79 quantity and quality of the DNA was determined 80 31 Microbiological media and growth on a 1.5% agarose gel and with a Nanodrop-1000 81 32 conditions Spectophotometer (Thermo Scientific, Wilmington, 82 DE). 83 33 Aliquots of 100 L from the solar-panel samples were 34 spread on Petri dishes containing LB medium (com- PCR amplification and 16S/18S rRNA 84 35 position in g/L: peptone 10.0, NaCl 10.0, yeast ex- massive sequencing 85 36 tract 5.0) or marine medium (composition in g/L: 37 peptone 5.0, yeast extract 1.0, ferric citrate 0.1, NaCl A set of primers adapted to massive sequencing for 86 38 19.45, MgCl2 5.9, Na2SO4 3.24, CaCl2 1.8, KCl 0.55, the Ion Torrent platform (Lifetechnologies) were used 87 39 NaHCO3 0.16, KBr 0.08, SrCl2 0.034, H3BO3 0.022, to capture 16S (modified from (7)) and 18S (modified 88 40 Na4O4Si 0.004, NaF 0.024, NH4NO3 0.0016 and from (8)) rRNA from the solar-panel DNA extraction 89 41 Na2HPO4 0.008), and incubated at room temper- in a PCR reaction. PCR reactions were performed 90 42 ature for 7 days. Individual colonies were indepen- with 30 ng of metagenomic DNA, 200 M of each of 91 43 dently re-streaked on new media and pure cultures the four deoxynucleoside triphosphates, 400 nM of 92 44 were finally identified through 16S rRNA sequencing each primer, 2.5 U of FastStart HiFi Polymerase, and 93 45 and crio-preserved in 20% glycerol (v/v) until re- the appropriate buffer with MgCl2 supplied by the 94 46 quired. A total of 53 bacterial strains were character- manufacturer (Roche, Mannheim, Germany), 4% of 95 47 ized under stress conditions and serially confronted 20 g/mL BSA (Sigma, Dorset, United Kingdom), and 96 48 with each other in solid medium to detect interactions 0.5 M Betaine (Sigma). Thermal cycling consisted 97 49 in terms of resistance to harsh conditions. of initial denaturation at 94C for 2 minutes followed 98 Page 2 of 13 bioRxiv preprint doi: https://doi.org/10.1101/029660; this version posted November 30, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 by 35 cycles of denaturation at 94C for 20 seconds, each coming from each of the directions on the 50 2 annealing at 50C for 30 seconds, and extension at paired-end sequencing. Those files were trimmed 51 3 72C for 5 minutes.

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