At many sites contaminated with hydrophobic organic chemicals such as polycyclic aromatic hydrocarbons (PAH), it has been observed that a fraction of pollutants sequesters with time, thus remaining unavailable for biodegradation. In addition, residual tar oils containing PAH are often dumped combined with small grain size waste coal and coke materials from processing at former gas work sites. In order to find suitable solutions for improving the biodegradation potential at the respective sites, predictions can be provided for the consequences of altered conditions for microbial growth and degradation. For optimisation of the remediation strategies and for interpreting observed effects, a combined model was applied for simultaneously considering dissolution from an organic chemical phase (non-aqueous phase liquids or solids), ad/desorption, sequestration (aging), microbial metabolism and growth, and the formation of non-extractable residues. The model has been verified from experimental observations in various aspects (Marchal et al. 2013, Kästner et al. 2014, Adam et al. 2014; see also presentation of Rein et al. on dissolution and microbial degradation of different PAH, at AquaConsoil 2015).
This model was used for the simulation of bioremediation options for clean-up of PAH-contaminated soils. The objectives were to understand the behaviour of PAH compounds in contaminated environments and to give recommendations for bioremediation measures based on this knowledge. We analysed the turnover of PAH by combining ad/desorption models for organic compounds with models for the growth and degradation kinetics of microbes. We modelled several scenarios and interpreted the observed effects, such as increasing distribution coefficient (Kd) and persistence of the PAH with time, decreasing degradation rates with concentration, and effects of amendments on sorption and degradation. Based on the kinetics of the processes and the fluxes in the system, we can provide a robust mathematical definition of the terms “bioavailability” and “bioaccessibility”. Finally, the model was applied to evaluate the most effective remediation strategy for PAH contaminated soils and sediments.
The modelling results indicate that added degrader bacteria only remove substrate for a short time-period. The addition of sorbents may decrease the bioavailable fraction. This results in lower plant uptake and toxicological risk, but may increase the persistent residual fraction. The persistence of a compound in aged soils can be overcome by increasing the desorption flux (e.g., by detergents or solvents e.g. acetone) and by stimulating bacterial growth by amendment with complex co-substrates (compost, root exudates). In addition, substrate affinity is an important factor for the competitiveness of bacteria in microbial communities under varying environmental conditions and in particular for varying available substrate abundances. It will thus finally determine the community structure at contaminated sites in general and particularly how the microbial community structure is affected by remediation measures.
Marchal G, Smith KE, Rein A, Winding A, Wollensen de Jonge L, Trapp S, Karlson UG. 2013. Impact of activated carbon, biochar and compost on the desorption and mineralization of phenanthrene in soil. Environ. Pollut. 181, 200-210.
Kästner M, Nowak KM, Miltner A, Trapp S, Schäffer A. 2014. Classification and modelling of non-extractable residue (NER) formation of xenobiotics in soil - a synthesis. Crit. Rev. Environ. Sci. Technol. 44 (19), 2107 - 2171.
Adam IKU, Rein A, Miltner A, Fulgêncio ACD, Trapp S, Kästner M. 2014. Experimental results and integrated modelling of bacterial growth on insoluble hydrophobic substrate (phenanthrene). Environ. Sci. Technol. 48 (15), 8717-8726.
Rein A, Adam IKU, Miltner A, Fulgêncio ACD, Brumme K, Trapp S, Kästner M. 2015. Impact of low-soluble hydrophobic substances on microbial turnover – Integrated modelling of microbial growth, degradation and dissolution from organic phase. Presentation at AquaConsoil 2015.
The microbial diversity linked to ETBE and TBA degradation in an ETBE-polluted aquifer was assessed by means of an integrated culture dependent/independent approach. Bench scale microcosm studies, isolation of ETBE/TBA degrading strains and microbiome analysis of native microbiota both in microcosms and in-situ stimulated groundwater were performed.
ETBE degrading strains were isolated directly from polluted groundwater (piezometer P11 and well W29) and from ETBE-degrading microcosms constructed from non-stimulated P11 groundwater. The obtained isolates were able to grow on mineral agar supplemented with vitamins under a saturated ETBE atmosphere as the sole source of carbon and energy. On the basis of colony morphology and 16S rRNA gene sequence, five strains were identified (ETBE-3 and ETBE-10 (belonging to Rhodococcus erytrhropolis), ETBE-8 (Sphingopyxis sp.), ETBE-11 (Hydrogenophaga pseudoflava) and ETBE-16 (Gordonia sp.) and selected for subsequent ETBE and TBA biodegradability assays. Three of the five strains (ETBE-3, ETBE-10 and ETBE-16) were able to degrade ETBE, whereas ETBE-8 (Sphingopyxis sp.) and ETBE-11 (H. pseudoflava) were only able to degrade TBA in batch assay conditions. The strain ETBE-16 was able to degrade both ETBE and TBA, but a transient accumulation of TBA was observed at early incubation stages. The synthetic consortium formed by the combination of the five strains was able to degrade 100 mg/L of ETBE in 5 days, without any transient accumulation of TBA. The co-culture of strains ETBE-16 and ETBE-11 was also able to prompt the complete ETBE biodegradation with no TBA occurrence during ETBE degradation.
In order to ascertain the environmental relevance of the isolated ETBE/TBA degrading strains, the microbial communities naturally occurring in groundwater samples and responding to microcosm incubations were analyzed by next generation sequencing (NGS). 16S rRNA gene-based 454-pyrosequencing from total Eubacteria was applied to i) initial groundwater (P11), and wells W23 and W29, before and after in-situ aerobic stimulation, and ii) microcosms from P11, which were aerobically stimulated with different N and P sources. The results showed that the microbial community structure from the groundwater (P11, W23 and W29) after the implementation of in-situ stimulation strategies was clearly different from the pre-existing microbiota. The predominant phylum in polluted groundwater prior to biostimulation (P11 and W23) corresponded to Proteobacteria with a relative abundance above 90% of the total population, being mainly composed by representatives of the Pseudomonadaceae family (Gamma-Proteobacteria). Members of the classes Alpha- and Beta-Proteobacteria, as well as from the phyla Bacteroidetes and Actinobacteria were also predominant in samples P11, W23 and W29 and in the microcosms. Multivariate correspondence analysis based on OTUs closely related to known ETBE/TBA degraders described in literature revealed a significant association of OTU 18 with ETBE exposed microcosms and non-stimulated groundwater (P11 and W23), but was not detected after in-situ biostimulation. Interestingly, OTU 18, accounting for 0.4% and 1% of the total community present in samples W23 and P11, respectively, was identical in sequence to the ETBE degrading isolates ETBE-3 and ETBE-10 and was highly homologous to the type strain of Rhodococcus erythropolis. After in-situ bioestimulation OTUs linked to other Actinobacteria, such as Arthrobacter spp. or Mycobacterium spp., became more predominant. In this sense, it is noteworthy that only one OTU closely related to Gordonia (strain ETBE-16) could be detected in W23 at late stages (t2) after in-situ biostimulation.
NGS results combined with microcosm studies and isolation of degrading strains revealed important shifts on the microbial community structure and the diversity of potential ETBE/TBA degraders affected by biostimulation strategies.
Coal tar, or creosote, as a by-product from coal gasification in municipal or manufactured gas plants (MGP), has been used for numerous industrial purposes including wood impregnation or preservation and as a raw material for a variety of commodity products. Being initially valued for its antibiotic properties, the constituents of coal tar, where released into soil and groundwater, now pose a considerable threat to soil and especially aquatic resources in many industrialized countries.
Coal tar is a complex mixture; its aromatic and phenolic constituents are considered the most worrisome from an environmental viewpoint. A comprehensive understanding of abiotic and enzymatically mediated pollutant transformation processes, involving complex contaminant (= tar oil), geochemical (= soil and aquifer mineral inventory) and microbial matrices (= mixed archaeal and bacterial communities), is required to control and, if desired, enhance biogeochemical reactions that contribute to the breakdown of organic contaminants.
In this context, one of the geochemical aspects less well understood is the role of poorly water soluble minerals that may serve as terminal electron acceptors (TEA) in anaerobic contaminant oxidation, including Fe(III) and Mn(IV) minerals, in hydrocarbon-contaminated aquifers. Despite their poor aqueous solubility and thus poor bio-accessibility, these mineral species participate in microbe-driven geochemical cycles. Several mechanisms, including electrochemically conductive pilus-like assemblages expressed by Geobacter species, or Shewanella’s chelating agents, enable for extracellular electron transport to practically insoluble Fe(III) and Mn(IV) surfaces serving as TEA in contaminant oxidation, have been ‘devised’ by nature.
The present study sets out to investigate artificial substitutes to these species-specific mechanisms to increase extracellular electron transfer processes connected to potential benefits for anaerobic contaminant oxidation. These extracellular electron shuttles (EES) are small organic and reversibly reducible/oxidizable structures that may participate in a large number of consecutive reduction and re-oxidation, driven by or in the absence of microbes. Both laboratory- and field-scale studies were performed.
Potential extracellular electron shuttles were characterized in laboratory trials using historically tar-oil contaminated soil and aquifers in terms of their efficiency to mediate the anaerobic oxidation of aromatic hydrocarbons, with a focus on EPA-PAH, N-, S- and O- substituted heterocyclic compounds as well as alkylated PAH. Contaminant transformation was monitored using GC-MS and comprehensive GCxGC-MS. The addition of various investigated structures, predominantly humic model substances (Quinones) in substoichiometric quantities were found to significantly increase biochemical and, to a lesser extent, abiotic contaminant oxidation in anaerobic bioreactors. A decrease in PAH concentrations was found to occur in parallel to an increase in aqueous-phase Mn and Fe-contents. These data suggest that using EES, a certain, soil or aquifer-specific poorly crystalline fraction of reducible Mn- and Fe-minerals are being made available to PAH-degrading organisms using EES. This fraction was predicted by incubating soil and aquifer samples with Shewanella alga and an easily accessible source of carbon and energy.
A small field trial was implemented in a microbially active, anaerobic tar oil contaminated aquifer with a high predicted Mn (IV) - availability. There, the increase in aqueous Mn- concentrations in the wake of the addition of a non-toxic EES into the groundwater points towards the possibility to stimulate electron transfer to poorly soluble geogenic terminal electron acceptors connected to the anaerobic oxidation of PAH. The analysis of ground water samples using GCxGC-TOF-MS revealed a qualitative change in hydrocarbon inventory and an enrichment in tentative PAH degradation products during EES application, suggesting a link of Mn- reduction to PAH oxidation facilitated by EES.
The application of extracellular electron shuttles to increase anaerobic oxidation processes is a direct approach to exploit indigenous, poorly soluble electron acceptors and thus represents an environmentally compatible bioremediation measure.
From a molecular level, geochemical and biogeochemical transformation processes involving hydrophobic contaminants and poorly accessible electron acceptors in complex matrices remain largely unexplored.
Methyl tertiary-butyl ether (MTBE) is a synthetic car fuel additive. However, it’s widely use resulted in groundwater contamination (up to 830 mg/L) with MTBE. Tert-butyl alcohol (TBA), an intermediate in MTBE degradation, is often found in association with MTBE contamination. Both compounds are very mobile in the subsurface and are threatening drinking water winning areas. They are, however, difficult to treat with existing pump and treat technologies (air stripping; sorption on activated carbon) due to their low sorption tendency, high water solubility and recalcitrancy. However, more efficient innovative technologies exist, which comprise biotechnology.
GROUNDWATER TREATMENT: Earlier, an aerobic MTBE/TBA-degrading bacterial consortium (M-consortium) has been isolated and its use for treating MTBE/TBA-contaminated groundwater was demonstrated. An inoculated bioreactor for ex-situ MTBE/TBA-removal from groundwater, as part of a pump and treat solution, was developed at lab-scale and demonstrated successfully at pilot scale. The technology realizes not only improved MTBE/TBA-removal, but is also more sustainable and eco-efficient in comparison with alternative methods, as the technology focuses on the destruction of the pollutants and not a relocation to other compartments (air, activated carbon).
Within the FP7 MINOTAURUS project (EU GA 265946) the robustness and reliability of biotechnologies like the inoculated bioreactor was investigated, which is important to come to innovation, being the full scale applications. Earlier, results from lab scale tests showed (1) that bioremediation is a valuable option to remove MTBE/TBA from groundwater and (2) that the M-consortium has potential as bacterial inoculum to enhance MTBE/TBA and BTEX-biodegradation under aerobic conditions. Next, pilot scale tests were performed to evaluate the robustness and reliability of the system at larger scale under real conditions. The results of 2 pilot test in the field will be summerized.
Test site 1: A pilot scale inoculated bioreactor (300L) was operated in the field at an industrial in Belgium for about 5 months. The prototype bioreactor was shown to be a relatively fast starting and stable system, removing MTBE (300-5000 µg/L) and TBA (3500-10000 µg/L) from the groundwater in an efficient way, hereby meeting regulatory limits.
Test site 2: An improved filling material to retain the biomass in the bioreactor was selected and used for the second bioreactor test. Firstly, the reactor was uploaded off-site where data indicated a good performance of the system. Next, the system was transported and operated at a petrol gas station (Belgium) treating groundwater contaminated with MTBE and TBA.
DRINKING WATER: The quality standards for drinking water are stricter in comparison with groundwater. Within the MIRESOWA-project (Danish project) different partners were active to evaluate the potential of biotechnology for treatment of polluted drinking water resources. The basis of the proposed technologies was bio-augmentation. Besides pesticides, MTBE and TBA were among the pollutants considered. At lab scale, bioreactors inoculated with the M-consortium were tested to remove MTBE and TBA at conditions that are inherent to drinking water production processes, being lower concentrations and higher fluxes. The results showed that the inoculated bioreactor technology offers potential for drinking water as it was possible to reduce the MTBE and TBA concentration below the regulatory limit (20 µg/L) without the need for frequent inoculation of the system.
Anaerobic bio-oxidation uses alternative electron acceptors instead of oxygen to destroy petroleum based contamination. This process occurs naturally at nearly all petroleum hydrocarbon contaminated sites. Cases with engineered addition of alternative electron acceptors are limited, as anaerobic oxidation is perceived as a slow process. However, the increased knowledge of bioremediation and recent studies have revealed that this process is more rapid than previously perceived, and that it has some significant advantages over the use of oxygen for bioremediation of petroleum hydrocarbons. In particular, the use of sulfate has gained recognition recently due to its high solubility and the limited potential for reactions with the soil matrix. Further, the absence of requirement for an energy intensive extraction and treatment system is an additional advantage.
An intensive study on the degradation capacity of sulfate on two site in Belgium, contaminated with BTEX, is described. Both the natural degradation capacity of soil and groundwater and the effect of addition of sulfate were evaluated. At one site a pilot test wit sulphate injection was performed and monitoring results of 1 year indicated a significant decrease of groundwater contamination.
For this evaluation, the following investigation was executed:
• Analysis of geochemical parameters to determine the geochemical conditions
• Sulfur and sulfate soil analysis were performed to estimate the quantity of reduced contamination and the quantity of sulfate available for biodegradation
• Biotraps® were used as microbial growth media in monitoring wells and analyzed after exposure to the aquifer. The following tests were undertaken:
o Biotraps® were marked with C-13 labeled benzene to evaluate the degradation velocity of benzene, the formation of CO2 and C-13 uptake in biomass.
o Biotraps® were analyzed on bacterial composition with polymerase chain reaction (PCR) analysis
o The above tests were performed under natural conditions and with sulfate addition
• Field pilottest with injection of sulphate and monitoring of degradation during one year.
Based on the findings, a remedial strategy with natural attenuation and focused application of sulfate in the source zones was selected and was preferred to a remedial strategy with a resource and energy-intensive multi-phase extraction.
The general principles of the technology and the investigation results of the case studies including the pilot test results will be presented.