'Biodeterioration of limestone: role of bacterial biofilms and possible intervention strategies' Philip Skipper (12255097) PhD Thesis June / 2018 1 To my wife, Lynda Skipper. Without whom none of this would have been possible. 2 Acknowledgements I would like to thank my supervisor, Doctor Ronald Dixon, for all his support, help, advice and encouragement, and for bravely allowing a slightly late topic change. I would also like to thank Doctor Ross Williams and Henning Schulze, my co-supervisors, for their advice and encouragement. Thanks also go to Michael Shaw for use of his 16S rRNA primers (p. 48, 49) as well as his friendship, to Alice Gillet for her friendship and provision of the surface profilometer work (p. 47, 66 & 67), and of course to the rest of the Microbiology laboratory team. I would also like to thank Doctor Lynda Skipper for her assistance in note taking and taking photographs during the initial sampling (p. 45, 46 & 60) as well as salt and biocide treatment, SEM imaging and EDX testing of the stone blocks for the salting analysis of biocides (p.53 & 163). I would like to thank Mary Webster for allowing me to use the isolates gathered during her Masters project and providing her sampling sheets (p. 45, 61 & Appendix A) and Doctor Alan McNally for PCR amplification and sequencing of the total population genomic DNA isolates for metagenomics and providing the raw data generated by the sequencer (p. 51 & 61). I am grateful to the School of Life Sciences, especially the former Head of School Professor Libby John, for covering the main research costs of this study. In addition I would like to thank Ronald Dixon for support for laboratory consumables from his gift fund and the College of Arts Research Resources Fund at the University of Lincoln which financially supported the initial sampling. This dissertation is the result of my own work and includes nothing that is the outcome of work done in collaboration except where it is specifically indicated in the text. 3 Abstract Limestone built heritage is at risk from the effects of biofilms, a microbial community encapsulated in a matrix of sugars, protein and extracellular DNA. Although biofilm research has been carried out in Mediterranean regions, few studies cover temperate Northern Europe climates, or the UK. This study concentrates on bacterial colonisation of Lincoln limestone, a highly vulnerable building material, and identifies the species, their role in biodeterioration and the efficacy of biocides against them. As part of this study the core species which comprise the bacterial component of the limestone microbiome have been characterised for the first time; this has allowed the identification of non- core species which are significantly associated with damaged and undamaged surfaces. Four mechanisms of biodeterioration have been identified, one previously unidentified, and isolated species have been characterised as to whether they are biodeteriorative and the mechanisms of biodeterioration that they employ. Two species, Curtobacterium flaccumfaciens and Solibacillus silvestris, have been characterised as producing biofilm matrix which actively causes biomechanical damage to the oolitic limestone structure as opposed to the passive enhancement of physical weathering which has been previously associated with biofilm matrix. Species capable of biodeterioration have also been shown to be present on both damaged and undamaged surfaces, something which has not been previously investigated. Environmental sampling, species identification and characterisation of species for biodeterioration have all combined to identify markers of biodeterioration, ie both physical markers and biomarkers. Specifically, a surface pH of 5.5 or lower and the presence of B. licheniformis is indicative of biodeterioration with a proportionally higher level of M. luteus when comparing damaged and undamaged stone. Finally this study brings the literature on conservation methods up to date by testing biocides which are in current usage, as many biocides in the literature are discontinued. This study is also the first in the field to show their efficacy against biofilm encapsulated bacteria and their propensity for chemically disrupting the biofilm matrix. 4 Table of Contents Acknowledgements 3 Abstract 4 Table of Figures 8 Professional development from this study 12 Research outputs from this study. 12 Training and Development 13 1 Introduction 14 1.1 Aims 14 1.2 Limestone 17 1.2.1 Lincolnshire limestones 18 1.2.2 The bioreceptivity of limestone 20 1.3 Limestone microbiome and its role in biodeterioration 22 1.3.1 The bacterial microbiome 24 1.3.2 Interpreting the bacterial microbiome 28 1.3.3 Biodeterioration 32 1.4 Conservation cleaning of limestone 36 1.4.1 Biocides 38 1.4.2 UVC light and cold plasma 41 1.4.3 Laser cleaning 42 1.4.4 Presure washing and steam cleaning 42 1.4.5 Selecting the appropriate method 43 2 Materials and Methods 44 2.1 Sampling 44 2.1.1 Sellotape and swab method to sample surfaces 44 2.1.2 Environmental data recording 44 2.1.3 Staining of sampled biofilms 46 2.1.4 Characterisation of Lincoln limestone 46 2.2 Characterisation of microorganisms 46 2.2.1 Culturing and isolation of bacteria 46 2.2.2 Identification of isolated species 47 2.2.3 Identification of micro-organisms – 16S rRNA sequencing with primer sequences. 47 2.2.4 Planktonic growth curves 48 2.2.5 pH profiling 49 5 2.2.6 Characterisation of biofilm formation 49 2.3 Metagenomics 50 2.4 Biocide testing 51 2.4.1 Agar spot test 51 2.4.2 Microdilution 52 2.4.3 Biofilm Calgary Peg method 52 2.4.4 Testing for potential enhancement of salt weathering by biocide 52 2.5 Testing for active dissolution of limestone 53 2.5.1 Effects of bacterial metabolism on the dissolution of calcium carbonate 53 2.5.2 pH modification of environment through growth curve. 53 2.5.3 SEM analysis of monoculture biofilms 54 2.6 Analysis of data 54 2.6.1 Analysis of metagenomic data 54 2.6.2 Statistical analysis of data 55 3 Sampling of the limestone microbiome 59 3.1 Introduction 59 3.2 Environmental sampling results 63 3.3 Physical characterisation of bioreceptivity 65 3.4 Identification of cultured isolates 69 3.5 Identification of species from metagenomic data 76 3.6 Analysis of the microbiome at the OTU level 78 3.6.1 Analysis of cultured isolates 78 3.6.2 Analysis of metagenomic isolates 81 3.7 Analysis of the microbiome at the Species level 87 3.7.1 Analysis of cultured isolates 87 3.7.2 Analysis of metagenomic isolates 90 3.7.3 Analysis of species common to both metagenomics and direct isolation 93 3.8 Discussion 95 3.8.1 Environmental measurements 95 3.8.2 Limestone as a habitable environment 96 3.8.3 The limestone bacterial microbiome 99 4 Characterisation of isolated bacteria 110 4.1 Introduction 110 4.2 Planktonic growth curves 111 6 4.3 Biofilm formation 114 4.4 Optimal growth pH 118 4.5 Metabolism of Industrial methylated spirits as a sole carbon source 123 4.6 Discussion 127 5 Bacterial biocorrosion of limestone 130 5.1 Introduction 130 5.2 pH modification of the environment. 130 5.3 Bacterial dissolution of calcium carbonate 136 5.4 SEM analysis of monoculture biofilms on limestone 137 5.5 Discussion 143 6 Biocide testing 148 6.1 Introduction 148 6.2 Spot plate testing of Microtech biocide and IMS 149 6.3 Biocide testing against bacteria growing planktonically. 151 6.4 Biocide testing against biofilm matrix and encapsulated bacteria 153 6.5 Analysis of salt deposition from biocides. 161 6.6 Discussion 165 7 Discussion 171 7.1 Conclusions 171 7.1.1 Bioreceptivity of Lincoln limestone 171 7.1.2 Microbiome 172 7.1.3 Biodeterioration 174 7.1.4 Biocides 178 7.2 Future work 178 References 183 Appendix A: Data from M. Webster Thesis 198 Appendix B: Köppen – Geiger definitions. 205 Appendix C: Species identified in metagenomic analysis 207 Appendix D: Species solely found in the rural environment. 217 7 Table of Figures Figure 1: Relative humidity for Lincolnshire, for the period from April 2012 to February 2016 with RH of 97% and higher indicated by the red dashed line. Chart produced using weatheronline.co.uk. Between October and February the RH regularly peaks over 97% RH with occasional peaks between March and June. .................................................................................... 20 Figure 2: Biofilm structure on a stone surface, the biofilm matrix consists of polysaccharides, extracellular DNA and proteins making up a structural matrix which the bacteria inhabit. Chemical gradients occur in the matrix with secretion of waste products such as nitric oxide and acids into the atmosphere and the stone surface. The matrix also provides protection against biocides. Diffusion of the waste products such as organic acids into the limestone results in weakening of the structure allowing increased penetration of the biofilm matrix into the stone. These mechanisms both result in the deterioration of the stone surface. Diagram by author and not to scale. ................................................................................................................................................. 23 Figure 3: Lincoln Cathedral between the Women’s Chapel and the Song School. Stonework in the foreground shows biological patina (dark stains) and damage, stonework in the background has been recently cleaned. On the left hand wall biological growth and spalling of the stone work are evident, especially where overhanging stonework provides a protective environment; this is especially clear at the bottom of the wall where there is a clear gradient between patinated stone and clean stone moving down from the overhang. On the right hand of the image, the Song School, water flow down the wall due to insufficient guttering, and shelter of the courtyard, has led to a damper environment than that on the left, patina is clearly evident working from the left near the door across towards the gutter downpipe where a combination of improved guttering and lead salts, which can be seen as a slight greying of the stone behind the downpipe itself, have led to a reduction in biological growth.