Paper mille genome project

Andrea Urso began his talk by describing Cartierre Carrara, the paper mill at which he is responsible for quality control, research and development. The paper mill has been producing high-quality tissue paper since 1873, for products including handkerchiefs, tablecloths, and napkins, across six production sites in Italy. Andrea explained that microbiota are introduced into paper via three routes: the environment, raw materials, and water. He highlighted how microbes vary greatly across different locations and environments: microbiota on cellulose pulp also vary by location across the world. He described the water cycle as the paper mill’s ‘gut’, where these microbes mix and proliferate: water is used across the full manufacturing process, from raw material to finished product. However, when it comes to bacterial analysis, 99% of strains cannot be cultured in the lab, as they require the specific environmental conditions of the industrial paper mill.

Andrea described how DNA sequencing is ‘the key’ to identifying the microbial species present through the paper manufacturing process. Andrea and his team developed a nanopore sequencing-based workflow to characterise these organisms: sequencing libraries are prepared using the Ligation Sequencing Kit and 16S Barcoding Kit, then sequenced on the MinION device. Data analysis is performed using the EPI2ME platform and custom programs generated in R. He noted that 16S PCR and Multiple Displacement Amplification techniques are also employed. Using the EPI2ME “What’s in my pot?” workflow, Andrea highlighted how 98% of the sequencing reads identified were of bacterial origin. The analysis revealed high levels of diversity: 2,955 bacterial species were identified in pulp and paper, whilst 7,747 were identified in water and the environment. Andrea and his team then developed a hierarchical clustering program in R to analyse the data.

In their first study, they analysed the raw materials used to make toilet paper, comprising short fibres (mostly eucalyptus) and large fibres (mostly pine), testing the hypothesis that similar bacteria would grow on similar wood. He showed a dendrogram revealing that microbial diversity clustering of the sequencing data successfully differentiated the short fibre pulp from the long fibre. Next, they wished to investigate whether the water cycle was providing a comfortable environment for some bacterial species. This revealed that, as well as microbes present from the pulp used, there were also microbes present that were suited to this environment and may be there for a long time. The microbes associated with pulp did not cluster with those suited to the water environment. They identified that the main species present in the water inlet were Cyanobacteria, common for this specific environment: water extracted from a well in an agricultural environment. These do not proliferate in the water cycle thanks to their chemical treatment protocol.

Finally, Andrea highlighted three field cases: ‘the green spots, the brown spots, and the smelling paper.’ Green spots are found on parts of the mill’s machinery; their analysis suggested that these are of environmental origin, from outside the mill, as they mostly comprise photosynthetic soil bacteria. This information then informed their cleaning strategy. Brown spots, of unknown origin, were found on paper: analysis of these spots compared with a clean paper control revealed a triple in the concentration of microbiota and their composition, which traced their origin to within the machine. Lastly, analysis of smelling paper revealed a microbial composition that clustered with the water cycle. These analyses have enabled Andrea and his team to develop a set of key performance indicators, based on the presence and abundance of specific bacteria, which are useful for routine control of the manufacture process.

Authors: Andrea Urso