Biofilm heterogeneity has been characterized on various scales for both natural and engineered ecosystems. This heterogeneity has been attributed to spatial differences in environmental factors. Understanding their impact on localized biofilm heterogeneity in building plumbing systems is important for both management and representative sampling strategies. We assessed heterogeneity within the confined engineered ecosystem of a shower hose by high-resolution sampling (200 individual biofilm sections per hose) on varying scales (μm to m). We postulated that a biofilm grown on a single material under uniform conditions should be homogeneous in its structure, bacterial numbers, and community composition. A biofilm grown for 12 months under controlled laboratory conditions, showed homogeneity on large-scale. However, some small-scale heterogeneity was clearly observed. For example, biofilm thickness of cm-sections varied up to 4-fold, total cell concentrations (TCC) 3-fold, and relative abundance of dominant taxa up to 5-fold. A biofilm grown under real (i.e., uncontrolled) use conditions developed considerably more heterogeneity in all variables which was attributed to more discontinuity in environmental conditions. Interestingly, biofilm communities from both hoses showed comparably low diversity, with <400 taxa each, and only three taxa accounting for 57%, respectively, 73% of the community. This low diversity was attributed to a strong selective pressure, originating in migrating carbon from the flexible hoses as major carbon source. High-resolution sampling strategy enabled detailed analysis of spatial heterogeneity within an individual drinking water biofilm. This study gives insight into biofilm structure and community composition on cm-to m-scale and is useful for decision-making on sampling strategies in biofilm research and monitoring.

Bath toys pose an interesting link between flexible plastic materials, potable water, external microbial and nutrient contamination, and potentially vulnerable end-users. Here, we characterized biofilm communities inside 19 bath toys used under real conditions. In addition, some determinants for biofilm formation were assessed, using six identical bath toys under controlled conditions with either clean water prior to bathing or dirty water after bathing. All examined bath toys revealed notable biofilms on their inner surface, with average total bacterial numbers of 5.5 × 106 cells/cm2 (clean water controls), 9.5 × 106 cells/cm2 (real bath toys), and 7.3 × 107 cells/cm2 (dirty water controls). Bacterial community compositions were diverse, showing many rare taxa in real bath toys and rather distinct communities in control bath toys, with a noticeable difference between clean and dirty water control biofilms. Fungi were identified in 58% of all real bath toys and in all dirty water control toys. Based on the comparison of clean water and dirty water control bath toys, we argue that bath toy biofilms are influenced by (1) the organic carbon leaching from the flexible plastic material, (2) the chemical and biological tap water quality, (3) additional nutrients from care products and human body fluids in the bath water, as well as, (4) additional bacteria from dirt and/or the end-users’ microbiome. The present study gives a detailed characterization of bath toy biofilms and a better understanding of determinants for biofilm formation and development in systems comprising plastic materials in contact with potable water.

26Upon entering building plumbing systems, drinking water bacteria experience 27 considerable changes in environmental conditions. For example, some flexible 28 polymeric materials leach organic carbon, which increases bacterial growth and 29 reduces diversity. Here we show that the carbon supply by a flexible polymeric material 30 drives nutrient-based selection within establishing biofilm communities. We found that 31 migrating carbon from EPDM coupons resulted in considerable growth for different 32 drinking water communities (0.2 -3.3 x 10 8 cells/cm 2 ). All established biofilm 33 communities showed low diversity (29 -50 taxa/biofilm), with communities dominated 34 by even viewer taxa (e.g., 5 taxa accounting for 94 ± 5 % relative abundance, n = 15). 35Interestingly, biofilm communities shared some taxa (e.g., Methylobacterium spp.) and 36 families (e.g., Comamonadaceae), despite the difference in starting communities. 37Moreover, selected biofilm communities performed better than their original 38 communities regarding maximum attachment (91 ± 5 vs. 69 ± 23 %, n = 15) and 39 attachment rate (5.0 ± 1.7 x 10 4 vs. 2.4 ± 1.2 x 10 4 cells/cm 2 /h, n = 15) when exposed 40 to new EPDM coupons. Our results demonstrate nutrient-based selection during initial 41 biofilm formation on a flexible polymeric material and a resulting benefit to selected 42 communities. We anticipate our findings to help connecting observational 43 microbiological findings with their underlying ecological principles. Regarding initial 44 biofilm formation, attachment dynamics, growth, and selection thereof are important 45 for the management of microbial communities. In fact, managing initial colonization by 46 supplying specific carbon and/or introducing consciously chosen/designed 47 communities potentially paves the way for a probiotic approach for building plumbing 48 materials. 49 50