Microorganisms and nanoparticles

NANOMICRO is a bioremediation project for soils involving the injection of exogenous microorganisms that are capable of feeding on the pollution present in these soils. Substrates enabling the correct development of exogenous and endogenous microrganisms (c-to-d present in polluted soils) are injected at the same time as the exogenous microorganisms. The original idea for the NANOMICRO project is to use nanoparticles to stimulate the growth and activity of exogenous microrganisms.

In order to understand the progress that has been made in terms of the depollution of soils, it is neccessary to take a look at the pollution itself. Pollution is a major problem because soils keep chemical products in their “memory”: organic pollutants such as dioxins, PCBs (polychlorobiphenyls) or PAHs (polycyclic aromatic hydrocarbons) as well as high levels of heavy metals such as lead, fertilizers, insecticides, antifungal products as well as weed-killers affect soils. Soils are an essential element of biodiversity because they contain 80% of the entire biomass. In the light of these facts and the urgent need to preserve these spaces, the European Commission launched a thematic strategy for the protection of soils which was adopted in 2007. Other local initiatives have also been initiated. In Wallonia, a decree relating to the remediation of soils has been in existence since 2004 completed by another decree dating from the end of 2008 relating to soil management, sets out a basis for the coordinated treatment of potentially polluted soils, while previously, the legislation only covered two kinds of sites: dumps and service stations. In addition, increased funding has been allotted to the restoration of sites earmarked for redevelopment (SAR) in the context of the Marshall Plan. It is in this context that the project NANOMICRO is to be seen, financed jointly by the Wallonia Regional Government and two industrial companies SANIFOX (coordinator of the project) and Artechno(4). The academic partners to the project are the Wallonia Centre of Industrial Biology(5) and the Department of Chemical Engineering – Nanomaterials Catalysis and Electrochemistry to which Stéphanie Lambert-Jamoulle, who is a research associate at the FNRS and also an assistant lecturer, belongs. These two research laboratories belong to the University of Liege. The NANOMICRO project was launched in January 2013 for a duration of 4 years. It aims to study and test the interaction between nanoparticles and microorganisms and their impact on the depollution of aromatic hydrocarbons in soils. The first results are just beginning to be published (6).

Participating in the perfection of bioremediation techniques

“ The basis of the project is to carry out what is called the bioremediation of soils which involves injecting polluted soils with microorganisms (in this case called exogenous microorganisms) capable of feeding on the pollution that is present in the soils. Substrates enabling the correct development of exogenous and endogenous microrganisms (c-to-d present in polluted soils) are injected at the same time as the exogenous microorganisms” explains Stéphanie Lambert-Jamoulle. The original idea for the NANOMICRO project was to use nanoparticles to stimulate the growth and activity of exogenous microorganisms. Bioremediation presents non-negligible advantages: reduced costs, total efficacy on volatile residual pollutants and on non-chlorinated pollutants such as lubricants, oils and a large number of aromatic hydrocarbons but also some chlorinated pollutants. In addition, it can be carried out in situ, requiring neither the excavation of soils nor heavy logistics particularly in terms of transport. It should be noted that the choice of treatments used is influenced by the intended future use of the terrain concerned. If it involves real estate operations, the time necessary before the remediation of the land is an essential parameter. In this case there will be little or no place for treatments in the long-term. The excavation and exportation of polluted soils are therefore chosen in the majority of cases. This solves the problem locally but raises the question of the treatment of the excavated soil. Bioremediation would seem to be the ideal solution in comparative terms. The main disadvantage lies in the duration of the treatment required, estimated to be about four years in Belgium for urgent situations and fifteen years in the other cases. This disadvantage is not to be taken lightly when we know the areas that need to be treated.

Thus, in 2012 a historical waste inventory began identifying the number of potentially polluted sites in Wallonia at between 3400 and 17000 according to whether there was a high or low-level of pollution, whereas 994 sites were counted where the level of pollution was established and 1322 were sites that had already been cleaned(7). It has now become clear that this is not just an environmental necessity but also a promising market from a commercial point of view. By way of example, it includes sites such as oil depots and major users of fuels (TEC and SNCB depots…), service stations, domestic tanks and non-excavatable urban sites. The company SANIFOX has been active for several years in the area of bioremediation-biostimulation (stimulation of endogenous and/or exogenous microorganisms) for the degradation of pollutants in soils and underground water. SANIFOX acts for leading clients in the chemical and petrochemical industry and among other activities targets the treatment of non-chlorinated pollutants, that is to say “the hydrocarbons you might find for example in the ground below a former petrol station”. NANOMICRO has therefore primarily focussed on these types of pollutants.

The beginnings of a success story

The NANOMICRO project is the result of an encounter between the Wallonia Centre for Industrial Biology (CWBI) directed at that time by Pr. Philippe Thonart and the company SANIFOX, which was co-founded by Benoît Lavigne who also happens to be the current manager. “The researchers at CWBI had succeeded in isolating and developing a strain of microorganisms that degrade aromatic hydrocarbons which was exactly what SANIFOX was researching in order to increase the kinetics of biodegradation, otherwise known as the speed of degradation of hydrocarbonated pollutants in soils which it treated for its clients. Moreover, in the wake of completely original and encouraging results in another area of biotechnology (the production of biohydrogen)(8) it was observed that the growth of the bacterial strain in question was stimulated when in the presence of nanoparticles which were synthesized in the Department of Chemical Engineering – Nanomaterials, Catalysis and Electrochemistry, explains Stéphanie Lambert-Jamoulle. This gave rise to the NANOMICRO project. In time, we would like to develop a microorganism/nanoparticle formula which could be used by the company SANIFOX in the context of soils treatments and polluted groundwater tables for its different clients. ” The particularity of the project lies in the strain of microorganisms on one hand and the procedure for the use of the nanoparticles on the other. In fact, the strain in question, Rhodococcus erythropolis, is the property of ARTECHNO and CWBI. In this case, the nanoparticles, with  sizes of 2-3 nm, present the specificity of being encapsulated in an inert silica matrix  which prevents them from sintering  and also being dispersed in a random fashion and uncontrolled in their natural state. The use of this specificity does not correspond to the method developed by other researchers which involves the use of metallic nanoparticles directly injected into the soil. In fact, there is an environmental risk with regard to the use of traditional nanoparticles (non-encapsulated) in bioremediation processes. It is linked to their high surface/volume ratio making them highly reactive which could lead to problems of toxicity. Stéphanie Lambert-Jamoulle explains: “A nanoparticle is a particle whose size is around 10⁻⁹ metres. It is extremely small and of course it is reactive because the smaller particles are, the more reactive they are due to the fact that the surface/volume ratio is very high. The danger with nanoparticles is that if they are liberated in an uncontrolled fashion, they risk polluting soils and runoff waters. ” In order to compensate for this, the nanoparticles have been “blocked” within another medium, inert silicone in this case. “ This is a micrometric powder, comparable in size to sand which can be found in the average garden. This silica powder is sufficiently porous to act as a reservoir for nanoparticles. We carried out different tests in the aqueous phase: when silica powder containing nanoparticles was added to water, almost none of these nanoparticles were re-released into the water after several weeks. ” Consequently, this was a great success!

Promising results and new challenges

The results of laboratory tests on the degradation of pollutants such as aromatic hydrocarbons showed immediately promising results. They then rose to new challenges concerning applications in real environmental conditions. In the laboratory, “we chose phenol and biphenyl-type pollutant molecules in order to be able to efficiently and rapidly determine the results of biodegradation over the course of time.” These pollutants are placed in an aerated aqueous phase ( oxygen in the ambient air is necessary for the growth of microorganisms, a little like fish in an aquarium) and is then put in contact with individual microorganisms or a mixture of microorganisms and nanoparticles encapsulated into  silica. “We observed the degradation over time and followed parameters such as temperature and pH, to achieve an extra 70% level of degradation of pollutants in 18 days when the nanoparticles accompany the microorganisms. In real environmental conditions, other parameters need to be taken into consideration. We noticed that when working in real conditions, the speed of degradation is much slower: the presence of inhibitors strongly influences the process of degradation and “uncoupling” the hydrocarbonated pollution from the soil matrix in order to make it accessible as a nutritive element for the microorganisms represents a sizeable challenge which we are actively working on to find original solutions”. The presence of compounds such as nitrates and chlorides that are sometimes present in polluted underground water can also affect the degradation reaction which can require a pre-treatment before beginning the degradation proper, so to speak. On the other hand, the results obtained were surprising for other families of pollutants but confidentiality is the order of the day.

In terms of prospects, the project, after four years of activity will certainly open the door to in-depth fundamental research on the exact impact of the exact interaction between microorganisms and nanoparticles of which very little is still known. Indeed, the nanoparticles in this case are encapsulated in a porous silica powder which means that they cannot enter into direct contact with the microorganism. One theory that has been offered is that the microorganism can secrete an enzyme which can interact with the nanoparticles to catalyze the speed of degradation of the aromatic compounds. The mechanisms involved in this need to be precisely understood. For the moment, the encounter between microorganisms and nanoparticles remains “an extraordinary aspect of the NANOMICRO project, which is both captivating and innovative at the same time”.

(1) Regional Government of Wallonia decree relative to the «remediation of polluted soils and active economic sites to be renovated » adopted on April 1st 2004
(2) Regional Government of Wallonia decree adopted on December 3rd 2008 and implemented on May 18th 2009.
(3) €243M have been allotted to the renovation of 37 polluted SARs (515 hectares) and €110 M to the renovation of 121 low-priority or slightly polluted SARs (258 hectares). In addition to this there is also the Marshall plan 2.vert which plans to provide €125M and €100M for the renovation of 13 polluted SARs and 70 non-polluted or slightly-polluted SARs. (Source : http : //etat.environnement.wallonie.be, tableau de bord 2010, p.     192)
(4) Artechno, created in 1999, is a spin-off-type company from research carried out at the Wallonia Centre for Industrial Biology.
(5) The Wallonia Centre for Industrial Biology develops research into biotechnology, from the initial stage to the finished product; it was jointly created in 1988 by the University of Liege and the University Faculty of Agronomic Sciences at Gembloux, today, Gembloux Agro-Bio Tech/University of Liege.
(6) Development by the sol–gel process of highly dispersed Ni–Cu/SiO2 xerogel catalysts for selective 1,2-dichloroethane hydrodechlorination into ethylene, Microporous and Mesoporous Materials, Pirard Sophie, Mahy Julien, Pirard Jean-Paul, Heinrichs Benoît, Raskinet Laurent, Lambert Stéphanie, ref. Orbi : http://hdl.handle.net/2268/172092
W. Wannoussa, T. Masy, S. D. Lambert, B. Heinrichs, L. Tasseroul, A. E. Al-Ahmad, F. Weekers, P. Thonart, S. Hiligsmann, Journal of Water Resource and Protection, 7 (2015) 264-277."Effect of iron nanoparticles synthesized by a sol-gel process on Rhodococcus erythropolis T902.1 for biphenyl degradation".
W. Wannoussa, S. Hiligsmann, T. Masy, S. D. Lambert, B. Heinrichs, A. E. Al-Ahmad, F. Weekers, P. Thonart, Journal of Sol-Gel Science and Technology, press article. "Effect of metal ions and metal nanoparticles encapsulated in porous silica on biodegradation kinetics for biphenyl".

(7) Source: Walloon Environment Portal. http://etat.environnement.wallonie.be/
(8) http://www.microh2.ulg.ac.be/, http://orbi.ulg.ac.be/handle/2268/143706

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