Bacterial nanocellulose production from naphthalene.

Polycyclic aromatic compounds (PAHs) are toxic compounds that are released in the environment as a consequence of industrial activities. The restoration of PAH-polluted sites considers the use of bacteria capable of degrading aromatic compounds to carbon dioxide and water. Here we characterize a new Xanthobacteraceae strain, Starkeya sp. strain N1B, previously isolated during enrichment under microaerophilic conditions, which is capable of using naphthalene crystals as the sole carbon source. The strain produced a structured biofilm when grown on naphthalene crystals, which had the shape of a half-sphere organized over the crystal. Scanning electron microscopy (SEM) and GC-MS analysis indicated that the biofilm was essentially made of cellulose, composed of several micron-long nanofibrils of 60 nm diameter. A cellulosic biofilm was also formed when the cells grew with glucose as the carbon source. Fourier transformed infrared spectroscopy (FTIR) confirmed that the polymer was type I cellulose in both cases, although the crystallinity of the material greatly depended on the carbon source used for growth. Using genome mining and mutant analysis, we identified the genetic complements required for the transformation of naphthalene into cellulose, which seemed to have been successively acquired through horizontal gene transfer. The capacity to develop the biofilm around the crystal was found to be dispensable for growth when naphthalene was used as the carbon source, suggesting that the function of this structure is more intricate than initially thought. This is the first example of the use of toxic aromatic hydrocarbons as the carbon source for bacterial cellulose production. Application of this capacity would allow the remediation of a PAH into such a value-added polymer with multiple biotechnological usages.

Occurrence and diversity of the oxidative hydroxyhydroquinone pathway for the anaerobic degradation of aromatic compounds in nitrate-reducing bacteria.

The nitrate-reducing betaproteobacteria Azoarcus anaerobius and Thauera aromatica AR-1 use an oxidative mechanism to anaerobically degrade resorcinol and 3,5-dihydroxybenzoate (3,5-DHB), respectively, rendering hydroxyhydroquinone as intermediate. The first pathway step is performed by a dimethylsulphoxide-reductase family hydroxylase. The gene cluster coding for the pathway is homologous in these strains. Only these two Rhodocyclales are known to follow this anaerobic pathway, and nothing is known about its distribution in prokaryotes. To determine the relevance and diversity of this strategy in nature, we enriched for bacteria able to oxidize resorcinol or 3,5-DHB under denitrifying conditions. Nitrate-reducing bacteria able to degrade these compounds were present in soil, aquifer and marine sediments. We were able to isolate a number of strains with this capacity from soil and aquifer samples. Amplicon libraries of rehL, the gene encoding the first step of this pathway, showed an overall low diversity, most sequences clustering with either pathway enzyme. Isolates belonging to the Beta- and Gammaproteobacteria able to grow on these substrates revealed rehL homologues only in strains belonging to Thauera and Azoarcus. Analysis of sequenced genomes in the databases detected the presence of highly similar clusters in two additional betaproteobacteria and in the gammaproteobacterium Sedimenticola selenatireducens, although anaerobic growth on a dihydroxyaromatic could only be confirmed in Thauera chlorobenzoica 3CB-1. The presence of mobile elements in the flanking sequences of some of the clusters suggested events of horizontal gene transfer, probably contributing to expand the pathway to a broader host range within the Proteobacteria.

Naphthalene biodegradation under oxygen-limiting conditions: community dynamics and the relevance of biofilm-forming capacity.

Toxic polycyclic aromatic hydrocarbons (PAHs) are frequently released into the environment from anthropogenic sources. PAH remediation strategies focus on biological processes mediated by bacteria. The availability of oxygen in polluted environments is often limited or absent, and only bacteria able to thrive in these conditions can be considered for bioremediation strategies. To identify bacterial strains able to degrade PAHs under oxygen-limiting conditions, we set up enrichment cultures from samples of an oil-polluted aquifer, using either anoxic or microaerophilic condition and with PAHs as the sole carbon source. Despite the presence of a significant community of nitrate-reducing bacteria, the initial community, which was dominated by Betaproteobacteria, was incapable of PAH degradation under strict anoxic conditions, although a clear shift in the structure of the community towards an increase in the Alphaproteobacteria (Sphingomonadaceae), Actinobacteria and an uncultured group of Acidobacteria was observed in the enrichments. In contrast, growth under microaerophilic conditions with naphthalene as the carbon source evidenced the development of a biofilm structure around the naphthalene crystal. The enrichment process selected two co-dominant groups which finally reached 97% of the bacterial communities: Variovorax spp. (54%, Betaproteobacteria) and Starkeya spp. (43%, Xanthobacteraceae). The two dominant populations were able to grow with naphthalene, although only Starkeya was able to reproduce the biofilm structure around the naphthalene crystal. The pathway for naphthalene degradation was identified, which included as essential steps dioxygenases with high affinity for oxygen, showing 99% identity with Xanthobacter polyaromaticivorans dbd cluster for PAH degradation. Our results suggest that the biofilm formation capacity of Starkeya provided a structure to allocate its cells at an appropriate distance from the toxic carbon source.

Polycyclic Aromatic Hydrocarbon-Induced Changes in Bacterial Community Structure under Anoxic Nitrate Reducing Conditions.

Although bacterial anaerobic degradation of mono-aromatic compounds has been characterized in depth, the degradation of polycyclic aromatic hydrocarbons (PAHs) such as naphthalene has only started to be understood in sulfate reducing bacteria, and little is known about the anaerobic degradation of PAHs in nitrate reducing bacteria. Starting from a series of environments which had suffered different degrees of hydrocarbon pollution, we used most probable number (MPN) enumeration to detect and quantify the presence of bacterial communities able to degrade several PAHs using nitrate as electron acceptor. We detected the presence of a substantial nitrate reducing community able to degrade naphthalene, 2-methylnaphthalene (2MN), and anthracene in some of the sites. With the aim of isolating strains able to degrade PAHs under denitrifying conditions, we set up a series of enrichment cultures with nitrate as terminal electron acceptor and PAHs as the only carbon source and followed the changes in the bacterial communities throughout the process. Results evidenced changes attributable to the imposed nitrate respiration regime, which in several samples were exacerbated in the presence of the PAHs. The presence of naphthalene or 2MN enriched the community in groups of uncultured and poorly characterized organisms, and notably in the Acidobacteria uncultured group iii1-8, which in some cases was only a minor component of the initial samples. Other phylotypes selected by PAHs in these conditions included Bacilli, which were enriched in naphthalene enrichments. Several nitrate reducing strains showing the capacity to grow on PAHs could be isolated on solid media, although the phenotype could not be reproduced in liquid cultures. Analysis of known PAH anaerobic degradation genes in the original samples and enrichment cultures did not reveal the presence of PAH-related nmsA-like sequences but confirmed the presence of bssA-like genes related to anaerobic toluene degradation. Altogether, our results suggest that PAH degradation by nitrate reducing bacteria may require the contribution of different strains, under culture conditions that still need to be defined.

The effect of oil spills on the bacterial diversity and catabolic function in coastal sediments: a case study on the Prestige oil spill.

The accident of the Prestige oil tanker in 2002 contaminated approximately 900 km of the coastline along the northern Spanish shore, as well as parts of Portugal and France coast, with a mixture of heavy crude oil consisting of polycyclic aromatic hydrocarbons, alkanes, asphaltenes and resins. The capacity of the autochthonous bacterial communities to respond to the oil spill was assessed indirectly by determining the hydrocarbon profiles of weathered oil samples collected along the shore, as well as through isotope ratios of seawater-dissolved CO2, and directly by analyses of denaturing gradient gel electrophoresis fingerprints and 16S rRNA gene libraries. Overall, the results evidenced biodegradation of crude oil components mediated by natural bacterial communities, with a bias towards lighter and less substituted compounds. The changes observed in the Proteobacteria, the most abundant phylum in marine sediments, were related to the metabolic profiles of the sediment. The presence of crude oil in the supratidal and intertidal zones increased the abundance of Alpha- and Gammaproteobacteria, dominated by the groups Sphingomonadaceae, Rhodobacteraceae and Chromatiales, whilst Gamma- and Deltaproteobacteria were more relevant in subtidal zones. The phylum Actinobacteria, and particularly the genus Rhodococcus, was a key player in the microbial response to the spill, especially in the degradation of the alkane fraction. The addition of inorganic fertilizers enhanced total biodegradation rates, suggesting that, in these environments, nutrients were insufficient to support significant growth after the huge increase in carbon sources, as evidenced in other spills. The presence of bacterial communities able to respond to a massive oil input in this area was consistent with the important history of pollution of the region by crude oil.