Novel nonylphenol-degrading bacterial strains isolated from sewage sludge: Application in bioremediation of sludge.

Nonylphenol (NP) is an anthropogenic pollutant frequently found in sewage sludge due to the insufficient degrading effectiveness of conventional WWTPs and has attracted attention as an endocrine disruptor. The aim of this study was to isolate specific NP-degrading bacteria from sewage sludge to be used in the degradation of this contaminant through bioaugmentation processes in aqueous solution and sewage sludge. Up to eight different bacterial strains were isolated, six of them not previously described as NP degraders. Bacillus safensis CN12 presented the best NP degradation in solution, and glucose used as an external carbon source increased its effect, reaching DT50 degradation values (time to decline to half the initial concentration of the pollutant) of only 0.9 days and a complete degradation in <7 days. Four NP metabolites were identified throughout the biodegradation process, showing higher toxicity than the parent contaminant. In sewage sludge suspensions, the endogenous microbiota was capable of partially degrading NP, but a part remained adsorbed as bound residue. Bioaugmentation was used for the first time to remove NP from sewage sludge to obtain more environmentally friendly biosolids. However, B. safensis CN12 was not able to degrade NP due to its high adsorption on sludge, but the use of a cyclodextrin (HPBCD) as availability enhancer allowed us to extract NP and degrade it in solution. The addition of glucose as an external carbon source gave the best results since the metabolism of the sludge microbiota was activated, and HPBCD was able to remove NP from sewage sludge to the solution to be degraded by B. safensis CN12. These results indicate that B. safensis CN12 can be used to degrade NP in water and sewage sludge, but the method must be improved using consortia of B. safensis CN12 with other bacterial strains able to degrade the toxic metabolites produced.

Dissipation of a mix of priority PAHs in soils by using availability enhancers. Effect of aging and pollutant interactions.

A remediation strategy using three non-toxic availability enhancers (two cyclodextrins and a rhamnolipid biosurfactant) was applied to various soils artificially contaminated with a mix of Polycyclic Aromatic Hydrocarbons (PAHs) considered priority pollutants at two levels of contamination: only with 7 low molecular weight PAHs (LMW PAHs, 5 with 3-ring and 2 with 4-ring - fluoranthene and pyrene) or with 14 PAHs (from 3 to 6 rings). Natural attenuation of PAHs in all soils showed degradation capacity for the LMW PAHs, with a final content of LMW PAHs <5% of their initial concentration. Conversely, the rest of PAHs (high molecular weight PAHs, HMW) remained in the soils (61% - 83.5%), indicating abiotic dissipation of HMW PAHs due to formation of non-extractable residues in soils. The influence of the presence of HMW PAHs on the degradation of the 7 LMW PAHs was also tested, showing a general decrease in the time to obtain 50% dissipation (DT50), statistically significant for acenaphthene, acenaphthylene and fluorene. Availability enhancers showed different effects on PAHs dissipation. 2-hydroxypropyl-ß-cyclodextrin (HP) decreased DT50 of some of the lighter PAHs, whereas the rhamnolipid (RL) caused a slight DT50 increase due to its initial toxicity on native soil microorganisms, but showing later high degradation rate for LMW PAHs. On the contrary, randomly methylated-ß-cyclodextrin (RAMEB) slowed down PAHs degradation due to its high adsorption onto soil surface, blocking the desorption of PAHs from the soils. The high number of experimental factors not studied simultaneously before (soil type, co-contamination, availability enhancers and incubation time) allowed to conduct a statistical analysis which supported the conclusions reached. Principal Component Analysis separated the studied PAHs in 3 groups, in relation with their molecular weight and Kow. The first principal component was related with LMW PAHs, and separate the inefficient RAMEB from the other availability enhancers.

Extraction of nonylphenol, pyrene and phenanthrene from sewage sludge and composted biosolids by cyclodextrins and rhamnolipids.

Sewage sludge generated by Waste Water Treatment Plants (WWTPs) are frequently used as organic amendments in agriculture, but they contain pollutants such as Potentially Toxic Elements (PTEs) and organic contaminants which contaminate the agricultural soils. The study presented here is part of a larger study based on the application of environmentally friendly chemical and biological techniques to decrease the content of organic pollutants in sewage sludge before agricultural application. The aim of this study was to evaluate the performance of biodegradable extractants, such as some cyclodextrins (CDs), ß-cyclodextrin (BCD), hydroxypropyl-ß-cyclodextrin (HPBCD) and randomly methylated-ß-cyclodextrin (RAMEB), and a biosurfactant (rhamnolipid, RL) on the removal and availability of pyrene (PYR), phenanthrene (PHE) and nonylphenol (NP) from several biosolids samples in order to improve their subsequent biodegradation. The influence of pollutants retention time on biosolids was studied, as well as the effect of each extractant on PTEs solubilization. Results obtained were pollutant and extractant-dependent. BCD extracted similar amounts of pollutants compared to water, whereas HPBCD and RAMEB actually increased the availability of the three pollutants in most of the samples and aging times. RL seems to be the best election for Polycyclic Aromatic Hydrocarbons (PAHs) extraction from biosolids, with percentages of extraction multiplied by more than 80 and 40 times for PHE and PYR, respectively, relative to water extraction. The extraction enhancement was the highest for NP, the most hydrophobic pollutant, reaching more than 500-fold higher with HPBCD and RAMEB. PTEs extractability was not affected by the different CDs used, but RL caused an increment in their soluble content what could endanger a subsequent biodegradation of the organic pollutants.

Natural and assisted dissipation of polycyclic aromatic hydrocarbons in a long-term co-contaminated soil with creosote and potentially toxic elements.

An enhanced bioremediation strategy was applied to an industrial soil co-contaminated with Polycyclic Aromatic Hydrocarbons (PAHs) and Potentially Toxic Elements (PTEs). Hydroxypropyl-ß-cyclodextrin (HPBCD) and a natural mixture of two rhamnolipids (RL) were added to increase PAHs bioavailability, and combined with a microbial consortium (MC) to biodegrade soil PAHs. Bioavailability of only six PAHs (3-, 4-ring PAHs) increased when using HPBCD, with a maximum increase about 2.8-fold higher. The highest dose of HPBCD (5%) enhanced PAH degradation, with the best results for 4-ring PAHs with treatments of HPBCD + MC (up to 48% degradation for pyrene and 43% for fluoranthene), whereas dissipation for 5-ring PAHs was very low and for 6-ring was negligible. The use of RL increased the bioavailability of 13 of the 16 PAHs studied, reaching up to 60-fold higher values for phenanthrene or 18-fold higher for acenaphtene. RL addition did not show degradation improvement in any situation, and even inhibited the scarce degradation observed in the control treatment. The high increase in availability of both PAHs and mainly PTEs when using RL as amendment could make them toxic for microorganisms. In fact, Microtox Acute Toxicity test using Aliivibrio fischeri and the absence of colony forming units (CFUs) of indigenous bacteria demonstrated the extremely high levels of toxicity in RL treated soil.

Combined use of microbial consortia isolated from different agricultural soils and cyclodextrin as a bioremediation technique for herbicide contaminated soils.

The phenylurea herbicide diuron is persistent in soil, water and groundwater and is considered to be a highly toxic molecule. The principal product of its biodegradation, 3,4-dichloroaniline, exhibits greater toxicity than diuron and is persistent in the environment. Five diuron degrading microbial consortia (C1C5), isolated from different agricultural soils, were investigated for diuron mineralization activity. The C2 consortium was able to mineralize 81.6% of the diuron in solution, while consortium C3 was only able to mineralize 22.9%. Isolated consortia were also tested in soil slurries and in all cases, except consortium C4, DT50 (the time required for the diuron concentration to decline to half of its initial value) was drastically reduced, from 700 days (non-inoculated control) to 546, 351, and 171 days for the consortia C5, C2, and C1, respectively. In order to test the effectiveness of the isolated consortium C1 in a more realistic scenario, soil diuron mineralization assays were performed under static conditions (40% of the soil water-holding capacity). A significant enhancement of diuron mineralization was observed after C1 inoculation, with 23.2% of the herbicide being mineralized in comparison to 13.1% for the control experiment. Hydroxypropyl-ß-cyclodextrin, a biodegradable organic enhancer of pollutant bioavailability, used in combination with C1 bioaugmentation in static conditions, resulted in a significant decrease in the DT50 (214 days; 881 days, control experiment). To the best of our knowledge, this is the first report of the use of soil-isolated microbial consortia in combination with cyclodextrins proposed as a bioremediation technique for pesticide contaminated soils.

Bioremediation of diuron contaminated soils by a novel degrading microbial consortium.

Diuron is a biologically active pollutant present in soil, water and sediments. It is persistent in soil, water and groundwater and slightly toxic to mammals and birds as well as moderately toxic to aquatic invertebrates. Its principal product of biodegradation, 3,4-dichloroaniline, exhibits a higher toxicity than diuron and is also persistent in the environment. On this basis, the objective of the study was to determine the potential capacity of a proposed novel diuron-degrading microbial consortium (DMC) for achieving not only diuron degradation, but its mineralisation both in solution as well as in soils with different properties. The consortium was tested in a soil solution where diuron was the only carbon source, and more than 98.8% of the diuron initially added was mineralised after only a few days. The consortium was composed of three diuron-degrading strains, Arthrobacter sulfonivorans, Variovorax soli and Advenella sp. JRO, the latter had been isolated in our laboratory from a highly contaminated industrial site. This work shows for the first time the potential capacity of a member of the genus Advenella to remediate pesticide-contaminated soils. However, neither of the three strains separately achieved mineralisation (ring-14C) of diuron in a mineral medium (MSM) with a trace nutrient solution (NS); combined in pairs, they mineralised 40% of diuron in solution, but the most relevant result was obtained in the presence of the three-member consortium, where complete diuron mineralisation was achieved after only a few days. In the presence of the investigated soils in suspension, the capacity of the consortium to mineralise diuron was evaluated, achieving mineralisation of a wide range of herbicides from 22.9 to 69.0%.

Natural attenuation of fluorene and pyrene in contaminated soils and assisted with hydroxypropyl-ß-cyclodextrin. Effect of co-contamination.

The objectives of this study were to investigate the mutual effect of the PAHs fluorene and pyrene on their respective biodegradation and dissipation processes in an agricultural soil, and to determine the effect of hydroxypropyl-ß-cyclodextrin (HPBCD), used to increase the bioavailability of PAHs, on such processes. Fluorene dissipation was primarily due to abiotic processes, although a small contribution from biodegradation was also observed. Therefore, fluorene dissipation did not increase with HPBCD and its presence did not significantly alter the dehydrogenase activity. In contrast to fluorene, pyrene dissipation depended primarily on biotic factors, with endogenous soil microorganisms capable of degrading pyrene, with large increases in dehydrogenase activity. HPBCD increased biodegradation rate of pyrene. The co-contamination of soil with both PAHs did not affect fluorene evolution, but significantly inhibited pyrene biodegradation. The different abilities of soil bacterial consortia to catabolize these PAHs are discussed. Additionally, the possibility that the abiotic loss of fluorene through volatilization had a significant effect on the microbial community biodegradation of both fluorene and pyrene is examined.