Rhamnolipid-enhanced solubilization and biodegradation of PAHs in soils after conventional bioremediation

The application of a rhamnolipid biosurfactant for enhanced solubilization and biodegradation of slowly desorbing polycyclic aromatic hydrocarbons (PAHs) in contaminated soils was determined in this study. The soil samples exhibited different levels of pollution and different bioremediation stages: the first soil originated from a creosote-polluted site, contained 4370 mg kg -1 of PAHs and had not been bioremediated; the second soil was the same as the first but had received bioremediation treatment with nutrient amendment in biopiles for a period of 5 months and contained 580 mg kg -1 of PAHs after this treatment; the third soil was treated by bioremediation for several years to reduce the concentration of PAHs to 275 mg kg -1. The kinetics of PAH desorption were determined to assess the magnitude of the slowly desorbing fractions present in the polluted soil and to optimize the biosurfactant effectiveness in terms of biodegradation. The soils that had been treated by bioremediation were enriched in slowly desorbing PAHs. The rhamnolipid at a concentration above its critical micelle concentration enhanced biodegradation in the soils that had been bioremediated previously. The measurement of residual concentrations of native PAHs showed the promoting effect of the biosurfactant on the biodegradation of the slowly desorbing fractions. Interestingly, benzo(a)pyrene was biodegraded in the soil that had been bioremediated for a long time. Rhamnolipid can constitute a valid alternative to chemical surfactants in promoting the biodegradation of slow-desorption PAHs, which is one of the most important problems in bioremediation, but the efficiency depends strongly on the bioremediation stage in which the biosurfactant is applied

Biotecnologia per a la recuperació sostenible de sòls contaminats

Soil is a living and life-giving non-renewable natural resource. Pollution associated with human activity is one of the main agents of soil degradation. Hydrocarbons are the most abundant group of soil pollutants, including the toxic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). Bioremediation, which utilizes the natural biodegradative capacities of soil microorganisms, is a sustainable technology with the potential to restore the natural functions of soil. Though its use in Europe has increased dramatically during the last decade, uncertainty regarding final end-point concentrations continues to hamper its widespread application. Laboratory biotreatability studies are a useful tool for designing and evaluating the potential of bioremediation strategies in the clean-up of specific sites. However, optimization of this biotechnology and the development of new diagnostic and monitoring tools require a comprehensive understanding of the metabolic microbial networks involved in pollutants removal. Metabolic studies with single bacterial cultures have proven essential for hypothesizing how microbial communities cooperate in the synergistic degradation of organic contaminants, with key populations initiating attacks to produce partially oxidized compounds that are then more efficiently mineralized by secondary degraders. Advances in molecular tools are not only facilitating more comprehensive analysis of culturable and non-culturable microbial populations, but also finer distinctions between active and non-active microorganisms, and quantification of the expression of key enzymatic functions. These innovations will help confirm and illuminate the actual role of previously hypothesized networks, revealing new microbial functions for exploitation

Bacterial benz(a)anthracene catabolic networks in contaminated soils and their modulation by other co-occurring HMW-PAHs

Polycyclic aromatic hydrocarbons (PAHs) are major environmental pollutants in a number of point source contaminated sites, where they are found embedded in complex mixtures containing different polyaromatic compounds. The application of bioremediation technologies is often constrained by unpredictable end-point concentrations enriched in recalcitrant high molecular weight (HMW)-PAHs. The aim of this study was to elucidate the microbial populations and potential interactions involved in the biodegradation of benz(a)anthracene (BaA) in PAH-contaminated soils. The combination of DNA stable isotope probing (DNA-SIP) and shotgun metagenomics of 13C-labeled DNA identified a member of the recently described genus Immundisolibacter as the key BaA-degrading population. Analysis of the corresponding metagenome assembled genome (MAG) revealed a highly conserved and unique genetic organization in this genus, including novel aromatic ring-hydroxylating dioxygenases (RHD). The influence of other HMW-PAHs on BaA degradation was ascertained in soil microcosms spiked with BaA and fluoranthene (FT), pyrene (PY) or chrysene (CHY) in binary mixtures. The co-occurrence of PAHs resulted in a significant delay in the removal of PAHs that were more resistant to biodegradation, and this delay was associated with relevant microbial interactions. Members of Immundisolibacter, associated with the biodegradation of BaA and CHY, were outcompeted by Sphingobium and Mycobacterium, triggered by the presence of FT and PY, respectively. Our findings highlight that interacting microbial populations modulate the fate of PAHs during the biodegradation of contaminant mixtures in soils

Inorganic carbon stimulates the metabolic routes related to the polyhydroxybutyrate production in a Synechocystis sp. strain (cyanobacteria) isolated from wastewater

Cyanobacteria are capable of transforming CO2 into polyhydroxybutyrate (PHB). In this study, different inorganic carbon concentrations (0–2 gC L−1) were evaluated for a Synechocystis sp. strain isolated from wastewater. Quantitative RT-qPCR was also performed to decipher the links between inorganic carbon and PHB and glycogen metabolism. 2 gC L−1 of bicarbonate stimulated cell growth, nutrients consumption and production of PHB. Using this concentration, a 14%dcw of PHB and an average productivity of 2.45 mgPHB L−1 d−1 were obtained. Gene expression analysis revelated that these conditions caused the overexpression of genes related to glycogen and PHB synthesis. Moreover, a positive correlation between the genes codifying for the glycogen phosphorylase, the acetyl-CoA reductase and the poly(3-hydroxyalkanoate) polymerase was found, meaning that PHB synthesis and glycogen catabolism are strongly related. These results provide an exhaustive evaluation of the effect of carbon on the PHB production and cyanobacterial metabolism

Polyhydroxybutyrate and glycogen production in photobioreactors inoculated with wastewater borne cyanobacteria monocultures

The aim of this study was to investigate the PHB and glycogen accumulation dynamics in two photobioreactors inoculated with different monocultures of wastewater-borne cyanobacteria, using a three-stage feeding strategy (growth phase, feast-famine phase and feast phase). Two cyanobacterial monocultures containing members of Synechocystis sp. or Synechococcus sp. were collected from treated wastewater and inoculated in lab-scale photobioreactors to evaluate the PHB and glycogen accumulation. A third photobioreactor with a complex microbial community grown in real wastewater was also set up. During each experimental phase different concentrations of inorganic carbon were applied to the cultures, these shifts allowed to discern the accumulation mechanism of carbon storage polymers (PHB and glycogen) in cyanobacteria. Conversion of one into the other was directly related to the carbon content. The highest PHB and glycogen contents (5.04%dcw and 69%dcw, respectively) were achieved for Synechocystis sp

Multi-omic profiling of a newly isolated Oxy-PAH degrading specialist from PAH-contaminated soil reveals bacterial mechanisms to mitigate the risk posed by polar transformation products

Polar biotransformation products have been identified as causative agents for the eventual increase in genotoxicity observed after the bioremediation of PAH-contaminated soils.Their further biodegradation has been described under certain biostimulation conditions; however, the underlying microorganisms and mechanisms remain to be elucidated. 9,10-Anthraquinone (ANTQ), a transformation product from anthracene (ANT), is the most commonly detected oxygenated PAH (oxy-PAH) in contaminated soils. Sand-in-liquid microcosms inoculated with creosote-contaminated soil revealed the existence of a specialized ANTQ degrading community, and Sphingobium sp. AntQ-1 was isolated for its ability to grow on this oxy-PAH. Combining the metabolomic, genomic, and transcriptomic analyses of strain AntQ-1, we comprehensively reconstructed the ANTQ biodegradation pathway. Novel mechanisms for polyaromatic compound degradation were revealed, involving the cleavage of the central ring catalyzed by Baeyer−Villiger monooxygenases (BVMO). Abundance of strain AntQ-1 16S rRNA and its BVMO genes in the sandin-liquid microcosms correlated with maximum ANTQ biodegradation rates, supporting the environmental relevance of this mechanism. Our results demonstrate the existence of highly specialized microbial communities in contaminated soils responsible for processing oxy-PAHs accumulated by primary degraders. Also, they underscore the key role that BVMO may play as a detoxification mechanism to mitigate the risk posed by oxy-PAH formation during bioremediation of PAH-contaminated soils

Inorganic carbon stimulates the metabolic routes related to the polyhdroxybutyrate production in a Synechocystis sp. strain (cyanobacteria) isolated from wastewater

Cyanobacteria are capable of transforming CO2 into polyhydroxybutyrate (PHB). In this study, different inorganic carbon concentrations (0–2 gC L-1) were evaluated for a Synechocystis sp. strain isolated from wastewater. Quantitative RT-qPCR was also performed to decipher the links between inorganic carbon and PHB and glycogen metabolism. 2 gC L-1 of bicarbonate stimulated cell growth, nutrients consumption and production of PHB. Using this concentration, a 14%dcw of PHB and an average productivity of 2.45 mgPHB L-1 d-1 were obtained. Gene expression analysis revelated that these conditions caused the overexpression of genes related to glycogen and PHB synthesis. Moreover, a positive correlation between the genes codifying for the glycogen phosphorylase, the acetyl-CoA reductase and the poly(3-hydroxyalkanoate) polymerase was found, meaning that PHB synthesis and glycogen catabolism are strongly related. These results provide an exhaustive evaluation of the effect of carbon on the PHB production and cyanobacterial metabolism.Authors want to acknowledge the support received by the Spanish Ministry of Science, Innovation and Universities (MCIU), the Research National Agency (AEI), and the European Regional Development Fund (FEDER) [AL4BIO, RTI2018-099495-B-C21]. Estel Rueda thanks the Spanish Ministry of Education, Culture and Sport [FPU18/04941] for her grant. Joaquim Vila is a Serra Húnter Fellow (Generalitat de Catalunya). Rubén Díez-Montero would also like to thank the Spanish Ministry of Industry and Economy for his research grants [IJC2019-042069-I].Peer ReviewedPostprint (published version

Rhizosphere-enhanced biosurfactant action on slowly desorbing PAHs incontaminated soil

We studied how sunflower plants affect rhamnolipid biosurfactant mobilization of slowly desorbing fractions of polycyclic aromatic hydrocarbons (PAHs) in soil froma creosote-contaminated site. Desorption kinetics of 13 individual PAHs revealed that the soil contained initially up to 50% slowly desorbing fractions. A rhamnolipid biosurfactantwas applied to the soil at the completion of the sunflower cycle (75 days in greenhouse conditions). After this period, the PAHs that remained in the soil were mainly present in a slowly desorbing form as a result of the efficient biodegradation of fast-desorbing PAHs by native microbial populations. The rhamnolipid enhanced the bioavailable fraction of the remaining PAHs by up to 30%, as evidenced by a standardized desorption extraction with Tenax, but the enhancement occurredwith only planted soils. The enhanced bioavailability did not decrease residual PAH concentrations under greenhouse conditions, possibly due to ecophysiological limitations in the biodegradation process thatwere independent of the bioavailability. However, biodegradationwas enhanced during slurry treatment of greenhouse planted soils that received the biosurfactant. The addition of rhamnolipids caused a dramatic shift in the soil bacterial community structure, which was magnified in the presence of sunflower plants. The stimulated groups were identified as fast-growing and catabolically versatile bacteria. This new rhizosphere microbial biomass possibly interacted with the biosurfactant to facilitate intra-aggregate diffusion of PAHs, thus enhancing the kinetics of slow desorption. Our results show that the usually limited biosurfactant efficiency with contaminated field soils can be significantly enhanced by integrating the sunflower ontogenetic cycle into the bioremediation design

Temporal hydrochemical and microbial variations in microcosm experiments from sites contaminated with chloromethanes under biostimulation with lactic acid

The objective of our research is to identify the sequence of degradation processes leading to microbial speciation of microorganisms involved in degradation of CT and CF under natural attenuation and lactic acid biostimulation conditions. To this end, a comparative study of two types of microcosm experiments was carried out to analyze two scenarios: natural attenuation and lactic acid biostimulation. Experiments were carried out with water and sediment from a field site located at a petrochemical complex whose hydrochemical background inhibited the natural attenuation of carbon tetrachloride and chloroform. A significant result of our work was that these experiments allowed us to identify the CT abiotic degradation processes, among which the abiotic degradation induced by the biogenic activity of Dechlorosoma suillum should be noted. Although this is an abiotic degradation, the metabolism of this microorganism generates green rust precipitates, which in turn favor the abiotic reductive dechlorination of CT. Other relevant result was the identification of the biotic reductive dechlorination of CF by a bacterium of the Clostridiales order. This result presented the particularity that an apparent absence of isotopic fractionation was observed because a mixture of chloroform of different origins was produced. Our research showed that these processes were more efficient, in terms of faster degradation rates, when biostimulation with lactic acid was carried out. This biostimulation could therefore be an efficient remediation strategy at sites contaminated by chloromethanes, especially in cases where a complex pollution history results in a rich hydrochemical background that makes it difficult natural attenuation

Subsoil heterogeneities controlling porewater contaminant mass and microbial diversity at a site with a complex pollution history

This study seeks to improve our understanding of the conceptual model of pollutant transport and fate in cases of DNAPL contamination at sites with a complex contamination history. The study was carried out in an unconfined aquifer of alluvial fans in the Tarragona Petrochemical Complex (Spain). Two boreholes were drilled and continuous cores were recovered in order to carry out a detailed core description at centimeter scale and a comprehensive sampling of borehole cores. The biogeochemical heterogeneity at these sites is controlled by the conjunction of lithological, hydrochemical and microbiological heterogeneities. Biodegradation processes of contaminant compounds take place not only at the level of the dissolved fraction in the aquifer but also at the level of the fraction retained in the fine, less conductive materials as shown by the biodegradation haloes of parent and metabolite compounds. Sampling the low-conductivity levels also allowed us to identify compounds, e.g. BTEX, that are the remaining traces of the passage of old contaminant plumes whose sources no longer exist. This enabled us to describe past biogeochemical processes and to partially account for the processes occurring today. Transition zones, characterized by numerous textural changes, constitute ecotones whose biostimulation could be effective in promoting the acceleration of the remediation of the multiple pollution at these sites