bs_bs_banner Bacterial nanocellulose production from naphthalene Patricia Marın,1,† crystallinity of the material greatly depended on the Sophie Marie Martirani-Von Abercron,1,† carbon source used for growth. Using genome min- Leire Urbina,2 Daniel Pacheco-Sanchez, 1 ing and mutant analysis, we identified the genetic Mayra Alejandra Castaneda-Cata~ na,~ 1 Alona~ Retegi,2 complements required for the transformation of Arantxa Eceiza2 and Silvia Marques 1,* naphthalene into cellulose, which seemed to have 1Estacion Experimental del Zaidın, Department of been successively acquired through horizontal gene Environmental Protection, Consejo Superior de transfer. The capacity to develop the biofilm around Investigaciones Cientıficas, Calle Profesor Albareda, 1, the crystal was found to be dispensable for growth Granada, 18008, Spain. when naphthalene was used as the carbon source, 2Materials + Technologies Research Group (GMT), suggesting that the function of this structure is more Department of Chemical and Environmental Engineering, intricate than initially thought. This is the first exam- Faculty of Engineering of Gipuzkoa, University of the ple of the use of toxic aromatic hydrocarbons as the Basque Country, Pza Europa 1, Donostia-San carbon source for bacterial cellulose production. Sebastian, 20018, Spain. Application of this capacity would allow the remedia- tion of a PAH into such a value-added polymer with multiple biotechnological usages. Summary Polycyclic aromatic compounds (PAHs) are toxic Introduction compounds that are released in the environment as a consequence of industrial activities. The restora- Polycyclic aromatic hydrocarbons (PAHs) are toxic com- tion of PAH-polluted sites considers the use of bac- pounds with a marked stability and resistance to degra- teria capable of degrading aromatic compounds to dation, which results in long-term persistence in the carbon dioxide and water. Here we characterize a environment, thus causing serious social concern. They new Xanthobacteraceae strain, Starkeya sp. strain are released into the environment as a result of industrial N1B, previously isolated during enrichment under activity, although natural sources, such as hydrocarbon microaerophilic conditions, which is capable of seeps, significantly contribute to the presence of these using naphthalene crystals as the sole carbon aromatic compounds in nature. Sixteen PAHs, among source. The strain produced a structured biofilm which naphthalene, are among the US EPA priority pol- when grown on naphthalene crystals, which had the lutants for their toxicity and potential mutagenic activity. shape of a half-sphere organized over the crystal. Current remediation strategies for aromatic hydrocarbon- Scanning electron microscopy (SEM) and GC-MS polluted sites are based on the capacity of many bacte- analysis indicated that the biofilm was essentially ria to degrade natural and synthetic aromatic compounds made of cellulose, composed of several micron-long to CO2 and water. On this basis, in situ remediation nanofibrils of 60 nm diameter. A cellulosic biofilm strategies rely on either providing nutrients to promote was also formed when the cells grew with glucose growth of the autochthonous microbial populations (bios- as the carbon source. Fourier transformed infrared timulation) or the addition of microbes with proven spectroscopy (FTIR) confirmed that the polymer was capacity to degrade a target contaminant (bioaugmenta- type I cellulose in both cases, although the tion). Bacteria able to degrade PAHs under aerobic con- ditions are ubiquitous and have been extensively characterized (Wackett and Hershberger, 2001). It is well Received 31 December, 2018; revised 18 February, 2019; accepted established that the PAH biodegradation pathways 21 February, 2019. involve aromatic mono- and dioxygenases for ring acti- *For correspondence. E-mail [email protected]; Tel. +34 958 181 600; Fax + 34 958 129600. vation (hydroxylation) and ring cleavage as most rele- † These two authors contributed equally. vant players (Vaillancourt et al., 2006; Ullrich and Microbial Biotechnology(2019) 12(4),662–676 Hofrichter, 2007). Under aerobic conditions, the simplest doi:10.1111/1751-7915.13399 Funding Information PAH naphthalene undergoes a series of transformations This work was supported by the European Regional Development to render salicylate, which is further oxidized to CO2 and Fund FEDER and grants from the Spanish Ministry of Economy and water. The genes for the pathway are highly conserved Competitiveness (BIO2017-82242-R). ª 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Nanocellulose production from PAHs 663 and generally organized into two operons (Habe and benefits. In fact, in hydrocarbon-polluted sites, environ- Omori, 2003), as in the archetypal NAH7 plasmid: mental bacterial isolates often only degrade a narrow nahABFCED (from naphthalene to salicylate), and range of PAHs, and cooperative processes involving a nahGTHINLOMKJ (from salicylate to pyruvate and consortium of strains with complementary capacities are acetyl-CoA), in addition to a regulator, nahR (Yen and frequently observed (Bouchez et al., 1995; Wang et al., Gunsalus, 1982). 2016). However, the availability of oxygen is often limited in Biofilms also play a relevant role in hydrocarbon hydrocarbon-polluted sites. Stimulation of the autochtho- biodegradation by making these hydrophobic compounds nous aerobic microbial communities by the presence of more accessible to microorganisms (Johnsen et al., an external organic carbon source results in a high oxy- 2005; Heipieper et al., 2007; Espinosa-Urgel and gen demand for aerobic degradation processes in ini- Marques, 2017). Biofilm formation at the hydrocarbon– tially aerobic sites, causing rapid oxygen depletion. This water interface is believed to increase the flux of hydro- results in a decreasing oxygen concentration gradient carbons to the cells by diminishing the distance between and in the development of anoxic conditions (Lovley, the carbon source and the cell surface (Wick et al., 2003). Nitrate is generally the alternative electron accep- 2001; Wackett, 2015). Moreover, the EPSs in biofilms tor at the edge between oxic and anoxic conditions (and constitute an extracellular matrix that influences hydro- also close to the surface of submerged sediments). In carbon utilization (Ennouri et al., 2016). This extracellular the oxic/anoxic interface at the fringes of polluted sites, matrix generally includes proteins, nucleic acids and the oxygen availability is likely to fluctuate (Kurt et al., exopolysaccharides, and its role in different bacteria and 2016), and aerobic and nitrate-reducing metabolisms under different environmental conditions has been generally coexist at the boundaries of polluted sites, addressed (Hinsa et al., 2003; Chang et al., 2007; where microaerophilic conditions prevail (Wilson and Martınez-Gil et al., 2013). Different hypotheses have Bouwer, 1997; Yagi et al., 2010). Under microaerophilic been proposed to explain the advantages of biofilm conditions [defined as dissolved oxygen in the range of EPSs in hydrocarbon degradation: adsorption of the 0–2mglÀ1 (between 0% and 25% air saturation at hydrophobic substrates to the matrix, retention of 30°C)], minute amounts of oxygen suffice to promote the enzymes, location of intermediate products and biosur- aerobic degradation of hydrocarbons (Yerushalmi et al., factants close to the cell, and an increase in the cell 2002; Tancsics et al., 2013). Interestingly, in strongly adhesion properties have been suggested as possible reduced polluted sediments where an anaerobic metabo- mechanisms enhancing the diffusion of hydrocarbons lism is expected, genes for aromatic monooxygenases from the organic phase to the cell (Harms et al., 2010). (i.e. aerobic degradation pathways) have been shown to Accordingly, a number of hydrocarbonoclastic bacteria be highly abundant and no increase towards the oxi- have been shown to develop biofilms when grown on dized plume leading edges has been observed, evidenc- hydrocarbon compounds as the sole carbon source ing that the niche partitioning between aerobic and (Golyshin et al., 2002; Mounier et al., 2014). In many anaerobic degraders in the field is not fully resolved (Lar- cases, the biofilm was only produced in the presence of entis et al., 2013). hydrophobic degradable substrates, whereas the loss of Polycyclic aromatic hydrocarbon degradation has gen- the biofilm forming capacity implied limited hydrocarbon erally been analysed in the laboratory with pure cultures degradation (Johnsen and Karlson, 2004; Klein et al., of microorganisms in the planktonic state. However, the 2008). Hydrocarbon degradation dependent on biofilm situation in the environment is quite different because formation has been observed both in real marine hydro- microbial processes usually involve biofilms composed carbon spill incidents, where biodegradation was of several coexisting bacterial groups that develop com- detected in bacterial flocs associated with oil droplets, plex self-organized supracellular structures, coordinated and in laboratory experiments (Hazen et al., 2010; Net- to create a suitable habitat for the community (Flemming zer et al., 2018). Nevertheless, the nature of the com- et al.,