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METODE DE

BIOREMEDIERE A SITE-

URILOR CONTAMINATE

DIN ROMANIA

Delia-Laura Popescua si Gheorghe Gamanecib

a Facultatea de Chimie, Universitatea din Bucuresti, Romania b Facultea de Inginerie, Universitatea Constantin Brancusi, Tg-Jiu, Gorj, Romania Rezumat Numarul site-urilor contaminate din Romania este foarte mare. Aceste terenuri prezinta o grava problema de mediu pe care Romania, ca tara membra al Uniunii Europene, trebuie sa o rezolve in urmatorii ani. De aceea sunt necesare noi si eficiente strategii si mijloace de monitorizare a acestor site-uri. De o importanta deosebita in rezolvarea acestor probleme de mediu sunt tehnologiile de remediere capabile sa combine o eficienta foarte inalta de decontaminare cu un impact asupra infrastructurii si organismelor vii scazut, precum si costuri foarte scazute. Aceste mijloace si tehnologii constau in combinarea a diferite aspecte de biologie, chimie si inginerie. De aceea, design-ul, dezvoltarea si managementul necesita o abordare interdisciplinara. Obiectivul principal al acestui articol este de a furniza o prezentare critica a aspectelor innovative ale tehnologiilor si mijloacelor de bioremediere folosite. Site-uri contaminate din judetul Gorj precum si masuri concrete de bioremediere vor fi prezentate.

APPROACHES TO THE

BIOREMEDIATION OF

CONTAMINATED SITES IN

ROMANIA

Delia-Laura Popescua and Gheorghe Gamanecib

a Faculty of Chemistry, University of Bucharest, Romania b Faculty of Engineering, Constantin Brancusi University, Tg-Jiu, Gorj, Romania Abstract Romania possesses a large number of contaminated lands. These sites constitute an enormous environmental problem that, as a recently European Union member, Romania has to take care of in the next few years. To address this issue, innovative and effective site-monitoring tools and strategies are necessary. Remediation technologies capable of combining high decontamination efficiency with low costs and impacts on the site infrastructure and living organisms represent a key point in solving the environmental problems. These tools and technologies consist of a combination of different aspects of biology, chemistry and engineering sciences. Therefore, their design, development, and management require a broad and interdisciplinary background. The main objective of this paper is to provide a critical overview of the innovative aspects of the bio-chemical and molecular microbiology tools and bioremediation technologies currently available on the market. The contaminated sites from coal industry in Gorj County, along with specific remediation measures will be presented herein.

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Introducere Poluarea apelor subterane si a solului reprezinta o problema globala ce poate conduce la acumularea de substante chimice in lantul trofic si poate afecta flora si fauna din habitaturile contaminate. Contaminarea resurselor subterane de apa cu diversi compusi organici reprezinta o problema de mediu semnificativa. Site-urile contaminate contin numerosi poluanti care prezinta un risc crescut pentru sanatatea oamenilor, animalelor si pentru mediul inconjurator. Desi in ultimii ani s-au inregistrat progrese substantiale in reducerea cantitatilor de emisii industriale, inca mai au loc importante emisii, se cunosc numeroase site-uri contaminate si in mod continuu se descopera altele noi [1]. Multe din aceste site-uri prezinta pericolul de a deveni surse de contaminare a rezervelor de apa potabila si, de aceea, constituie un substantial hazard pentru sanatatea generatiilor actuale si viitoare. Pentru a remedia aceasta situatie s-au dezvoltat numeroase tehnologii de remediere. In acest articol vom prezenta tehnologii de bioremediere. Bioremedierea este procesul in care microorganismele metabolizeaza contaminantii printr-un proces oxidant sau reducator [2]. In conditii favorabile, microorganismele pot degrada complet, oxidative, contaminanti pana la produsi netoxici cum ar fi CO2, H2O sau acizi organici si metan. Procesele de bioremediere pot fi conduse spre realizarea: (1) oxidarea completa a contaminantilor organici (numita mineralizare), (2) biotransformarea compusilor organici in metaboliti mai mici si mai putin toxici, sau (3) reducerea gruparilor electrofile, cum ar fi nitro-, prin transfer electronic de la un donor (in general zaharuri sau acizi grasi), obtinandu-se compusi mai putin toxici. Cresterea numarului de posibilitati de decontaminare reusite, remedierea

Introduction Pollution of groundwater and soil is a worldwide problem that can result in uptake and accumulation of toxic chemicals in food chains and harm the flora and fauna of affected habitats. The contamination of groundwater resources by organic chemicals is a significant environmental problem. Contaminated sites often contain numerous pollutants, which can constitute a risk to health of humans, animals and or the environment. Although substantial progress has been made in reducing industrial releases over recent years, major releases still occur; a considerable number of known polluted sites exist and new ones are continually being discovered [1]. Many of these sites threaten to become sources of contamination of drinking water supplies and thereby constitute a substantial health hazard for current and future generations. To remedy this situation, numerous remediation techniques have been developed. This paper is focused on bioremediation techniques. Bioremediation is a process in which microorganisms metabolize contaminants either through oxidative or reductive processes [2]. Under favorable conditions, microorganisms can oxidatively degrade organic contaminants completely into non-toxic by-products such as carbon dioxide and water or organic acids and methane. Bioremediation processes may be directed towards accomplishing: (1) complete oxidation of organic contaminants (termed mineralization), (2) biotransformation of organic chemicals into smaller less toxic metabolites, or (3) reduction of highly electrophilic halo- and nitro- groups by transferring electrons from an electron donor (typically a sugar or fatty acid) to the contaminant, resulting in a less toxic compound. With increasing numbers of successfully demonstrated cleanups, biological remediation alone or in combination with other methods, has gained an established place as a soil restoration

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biologica sau in combinatie cu alte metode, reprezinta una dintre cele mai utilizate metode pentru restaurarea solurilor contaminate. Modalitati de bioremediere Principalele tipuri de bioremediere sunt [3]: Biostimularea Nutrienti si oxigen in forma lichida sau gazoasa se adauga in apele sau solurile contaminate pentru a favoriza dezvoltarea si activitatea bacteriilor existente. Se monitorizeaza disparitia contaminantilor pentru a determina nivelul de remediere. Bioaugmentarea Microorganisme ce pot degrada anumiti contaminanti sunt introdusi in sol sau apa. Bioaugmentarea este folosita mai des si cu success pentru degradarea contaminantilor din instalatiile de purificare a apei. Pana astazi, aceasta metoda nu s-a dovedit foarte eficienta pentru decontaminarea locurilor in care conditiile de optima crestere a microorganismelor adaugate nu pot fi controlata. Bioremedierea intrinseca Se mai numeste si atenuare naturala si are loc cu precadere in mod natural in solurile si apele contaminate. Aceasta bioremediere naturala este datorata microorganismelor si are loc in site-urile contaminate cu petrol, cum ar fi la statiile vechi de alimentare cu carburanti. Folosirea acestei tehnici necesita o monitorizare foarte atenta pentru a proteja mediul si sanatatea oamenilor. Bioremedierea in prezenta aerului sau oxigenului se numeste bioremediere aeroba si are decurge printr-un proces de oxidare totala sau partiala prin care se obtin constituenti minerali: CO2 si H2O. In conditii anaerobe, procesele de bioremediere sunt mult mai complexe. Contaminantii organici pot fi mineralizatidaca in mediu se gasesc nitrati si sulfati. In bioremedierea metanogenica,

technology. Bioremediation Approaches The main types of bioremediation are as follows [3]: Biostimulation -- Nutrients and oxygen - in a liquid or gas form - are added to contaminated water or soil to encourage the growth and activity of bacteria already existing in the soil or water. The disappearance of contaminants is monitored to ensure that remediation occurs. Bioaugmentation -- Microorganisms that can clean up a particular contaminant are added to the contaminated soil or water. Bioaugmentation is more commonly and successfully used on contaminants removed from the original site, such as in municipal wastewater treatment facilities. To date, this method has not been very successful when done at the site of the contamination because it is difficult to control site conditions for the optimal growth of the microorganisms added. Scientists have yet to completely understand all the mechanisms involved in bioremediation, and organisms introduced into a foreign environment may have a hard time surviving. Intrinsic Bioremediation -- Also known as natural attenuation, this type of bioremediation occurs naturally in contaminated soil or water. This natural bioremediation is the work of microorganisms and is seen in petroleum contamination sites, such as old gas stations with leaky underground oil tanks. Researchers are studying whether intrinsic bioremediation happens in areas with other types of chemical contamination. Application of this technique requires close monitoring of contaminant degradation to ensure that environmental and human health are protected. Bioremediation in the presence of air or oxygen is called aerobic bioremediation and typically proceeds through oxidative processes to render the contaminant either

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contaminantii sunt convertiti in metan, CO2 si urme de hydrogen. Un alt tip de biodegradare anaeroba este dehalogenarea reductive, prin care contaminantii devin mai putin toxici in urma unui process de dehalogenare. In general, bioremedierea aeroba este mai rapida decat bioremedierea anaeroba, de aceea este adesea preferata. Totusi, multi compusi pot fi metabolizati doar in conditii reducatoare, iar atunci biodegradarea anaeroba este singura optiune. Bioremedierea poate fi realizata in-situ, se numeste bioremediere in-situ, sau ex-situ, numita bioremediere ex-situ [3]. Bioremedierea este folosita in mod current pentru tratarea solurilor si apelor subterane contaminate cu compusi organici. Microbii pot, de asemenea, transforma poluanti anorganici cum ar fi ammoniac, nitrati si perclorati. Desi microorganismele nu pot degrada metale grele, ei pot fi folositi pentru schimbarea starii de oxidare a metalelor ceea ce le transforma in forme immobile si mai putin toxice. De exemplu, microbii pot converti cromul hexavalent in crom trivalent [2]. Bioremedierea poate fi folosita pe orice tip de sol, atunci cand continutul de umiditate este adecvat. Singura problema este ca in cazul solurilor cu permeabilitate scazuta este dificil de realizat aportul de oxygen si nutrienti. Trebuie mentionat ca un continut ridicat de contaminanti poate fi toxic pentru microorganisme. De aceea, o investigatie de fezabilitate este necesara pentru a determina daca biodegradarea este o optiune viabila pentru solurile contaminate din site-uri specifice in conditiile date. Biochimia proceselor de biodegradare Microbii prezinta o diversitate metabolica. O consecinta a acestei diversitati este faptul ca multi compusi organici, toxici sau persistenti, de natura antropogena, pot fi degradati prin activitate microbiala. In termini simpli,

partially oxidized to less toxic by-products or fully oxidized to mineral constituents: carbon dioxide and water. Under anaerobic conditions, bioremediation processes are more complex. In anaerobic processes, organic contaminants can be mineralized provided sufficient nitrate or sulfate is present. In methanogenic bioremediation, the contaminants are converted to methane, carbon dioxide and traces of hydrogen. Another type of anaerobic bioremediation is reductive dehalogenation where the contaminants are rendered less toxic by removal of halogens such as chlorine or nitro groups. Typically, aerobic bioremediation is quicker than anaerobic bioremediation; therefore, it is often preferred. However, many compounds can only be metabolized under reductive conditions and therefore anaerobic treatment is the only option. Bioremediation can be accomplished under in-situ conditions, called in-situ bioremediation, or under ex-situ conditions, called ex-situ bioremediation [3]. Bioremediation is commonly used for the treatment of soils and groundwaters contaminated with organic contaminants. Microbes can also successfully transform some inorganic pollutants such as ammonia, nitrate, and perchlorate. Although microbes cannot degrade heavy metals, they can be used to change the valence states of these metals thus converting them into immobile or less toxic forms. For example, microbes can convert mobile hexavalent chromium into immobile and less toxic trivalent chromium [2]. Bioremediation can be used in any soil type with adequate moisture content, although it is difficult to supply oxygen and nutrients into low permeability soils. It should be noted that very high concentrations of the contaminants may be toxic to microorganisms and thus may not be treated by bioremediation. Therefore, a feasibility investigation is needed to determine if biodegradation is a viable

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microorganismele trebuie sa primeasca energie din transformarea contaminantilor pentru a putea supravietui. Mai mult, trebuie sa existe o sursa de carbon pentru a putea construi material cellular. In absenta acestora, biodegradarea nu are loc. Bioremedierea depinde de procesele biologice naturale ale microorganismelor, unul dintre acestea fiind metabolismul. Enzimele controleaza toate reactiile ce au loc in cellule. Acestea catalizeaza atat reactiile de oxidare cat si pe cele de reducere a compusilor organici pentru producerea de energie (numite reactii catabolice), precum si producerea de noi componente celulare in timpul cresterii (numite reactii anabolice). Degradarea oricarei molecule organice necesita producerea si utilizarea eficienta a enzimelor [4]. Microorganismele au nevoie de conditii de mediu adecvate pentru a supravietui si a se dezvolta. Aceste conditii include pH adecvat, temperatura, oxigen, nutrienti, precum si lipsa compusilor toxici sau inhibitori. In general, bioremedierea decurge cu eficienta maxima la pH aproape de 7. Totusi, bioremedierea se poate realiza in intervalul de pH 5.5 - 8.5. Cele mai multe sisteme de bioremediere opereaza la temperaturi cuprinse intre 15 C si 45 C. Microorganismele aerobe necesita o anumita cantitate de oxygen pentru supravietuire, dar si pentru medierea reactiilor chimice la care participa. In general, o concentratie a oxigenului mai mare de 2 mg/L este necesara pentru microorganismele aerobe pentru a degrada eficient poluantii organici. De asemenea, microorganismele au nevoie de nutrienti pentru crestere. Nutrientii principali contin carbon, hydrogen, oxygen, azot si fosfor si prezinta formula generala (C60H82O23N12P). Cantitatea necesara din acesti nutrienti depinde de consumul de oxygen biochimic (BOD) al solului contaminat. In general, raportul C la N la

option for the site-specific soil and contaminant conditions. Biochemistry of Biodegradation Microbes are known for their metabolic diversity. One consequence of this diversity is the fact that many toxic or persistent anthropogenic organic compounds are degraded by microbial activities. In simple terms, microorganisms must gain energy from the transformation of contaminants in order to survive. In addition, they must also have a source of carbon to build new cell material. Absent these, biodegradation will not proceed. Bioremediation depends on the natural biological processes of microorganisms, one of which is metabolism. Enzymes control all reactions in cells. Enzymes catalyze both the oxidation and reduction of organic compounds for energy (called catabolic reactions) as well the production of new cell components during growth (called anabolic reactions). The degradation of any organic molecule, thus, requires the production and efficient utilization of enzymes [4]. Microorganisms need appropriate environmental conditions to survive and grow. These conditions include appropriate pH, temperature, oxygen, nutrients, and lack of inhibiting or toxic compounds. Typically, bioremediation is most efficient at a pH near 7. However, bioremediation can be achieved between pH values of 5.5 and 8.5. Most bioremediation systems operate over a temperature range of 15 C to 45 C. Aerobic microorganisms need a certain amount of oxygen not only to survive, but also to mediate their reactions. Generally, oxygen concentration greater than 2 mg/L is required for aerobic microorganisms to efficiently degrade organic pollutants. Microorganisms need nutrients for their growth. The major nutrients needed are identified with the generalized biomass formula (C60H82O23N12P) and include carbon, hydrogen, oxygen, nitrogen and phosphorous. The actual quantity of these

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P (raport de masa) necesar este 120:10:1. Alti nutrienti, cum ar fi sodium, potasiu, amoniu, calciu, magneziu, fier, clor, sulf sunt necesari in cantitati mici, intre 1 si 100 mg/L. De asemenea sunt necesari, in cantitati infime (mai mici de 1 mg/L), nutrienti cum ar fi mangan, cobalt, nichel, vanadiu, bor, cupru, zinc, diverse substante organice (vitamine) si molibden. Substantele toxice saucu actiune inhibitoare trebuie indepartate din mediu, atunci cand este posibil. Concentratii ridicate din orice contaminant pot fi toxice pentru microorganisme. Anumiti contaminanti pot fi toxici pentru microbi chiar la concentratii scazute. De obicei, problemele de toxicitate se remediaza prin dilutie sau microbi aclimatizati. Este de dorit ca umiditatea solului sa se pastreze intre 40 si 80%. Diverse clase de poluanti organici prezinta scheme de degradare diferite si de aceea conditiile si strategiile de bioremediere difera [5]. Atunci cand nivelul nutrientilor, umiditatii si microorganismelor din sol este scazut, acestea pot fi adaugate in mediu [5]. De asemenea, pentru prevenirea si incetinirea contaminarii solului prin migrarea contaminantilor, se pot amplasa diverse materiale naturale sau de sinteza pentru delimitarea zonelor contaminate. Tratarea se realizeaza prin biodegradare, uneori in combinatie cu aerare si fotooxidare sub actiunea soarelui. Viteza acestor procese creste in conditii de caldura si umiditate. Procesele de biodegradare se incetinesc drastic in timpul lunilor de iarna, cand temperaturile sunt scazute si zapada acopera pamantul Advantajele si dezavantajele bioremediarii Bioremedierea prezinta urmatoarele avantaje [4]:

Poate conduce la completa degradare a compusilor organici in produsi netoxici,

nutrients depends on the biochemical oxygen demand (BOD) of the contaminated soil. Generally, the C to N to P ratio (by weight) required is 120:10:1. Other nutrients such as sodium, potassium, ammonium, calcium, magnesium, iron, chloride and sulfur are needed in minor quantities, in the concentration range of 1 to 100 mg/L. In addition, traces (less than 1 mg/L) of nutrients such as manganese, cobalt, nickel, vanadium, boron, copper, zinc, various organics (vitamins) and molybdenum are needed. One must be careful that toxic substances do not exist that will produce adverse conditions for bioremediation. High concentration of any contaminant can frequently be toxic to microbes. Some contaminants even at low concentrations may be toxic to microbes. Generally, toxicity concerns are addressed by dilution or acclimated microbes, or by induced bioavailability limitations. It is also desirable to maintain the soil moisture level between 40 to 80% of field capacity. Different classes of organic pollutants have different microbial degradation pathways and thus different considerations for bioremediation strategies [5]. Other chemicals that are important to a microorganism include chemical compounds in the phosphorus, potassium, calcium and sodium group. Microorganisms also need trace elements of other chemicals, including chromium, cobalt, copper, and iron, all of which can be available in abundance at contaminated sites. Microorganisms, nutrients, and moisture may also be added [5]. Clay or plastic liners may be installed in the field prior to placement of the contaminated soil, which act to retard or prevent migration of contaminants into underlying and adjacent clean soils, groundwater, and surface water. Treatment is achieved through biodegradation, in combination with aeration and possibly photo oxidation in sunlight. These processes are most active in warm, moist sunny conditions. Treatment is greatly diminished

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Necesita un minimum de echipament mecanic,

Poate fi implementata ata in procese in-situ, cat si ex-situ. Bioremedierea in-situ este mult mai putin periculoasa deoarece nu necesita excavarea solurilor contaminate si, de asemenea, nu afecteaza zonele naturale limitrofe site-ului,

Costurile associate sunt mai scazute comparativ cu cele aferente altor tehnologii de remediere.

Bioremedierea prezinta urmatoarele dezavantaje [4]:

Exista potentialul unei degradari partiale in metaboliti care pot prezenta toxicitate chiar mai ridicata,

Procesul este foarte susceptibil la conditiile de mediu si prezenta unor inhibitori,

Este necesara o monitorizare continua pentru determinarea vitezelor de biodegradare,

Este dificil de controlat prezenta compusilor organici volatili in timpul proceselor de bioremediere ex-situ,

In general, necesita un tratament de durata comparativ cu alte tehnologii de remediere.

Bibliografie [1] Anastas, P. T.; Warner, J. C., Green Chemistry: Theory and Practice, 1998, Oxford University Press Inc., New York. [2] Meagher, R.B. Current Opinion in Plant Biology, 2000, 3 (2): 153-162. [3] Diaz E (editor), Microbial Biodegradation: Genomics and Molecular Biology, 1st ed., Caister Academic Press, 2008 [4]

or even completely arrested during winter months when temperatures are cold and snow covers the ground. Advantages and Disadvantages of Bioremediation Bioremediation has the following advantages [4]:

It may result in complete degradation of organic compounds to nontoxic byproducts,

There are minimum mechanical equipment requirements,

It can be implemented as in-situ or ex-situ process. In-situ bioremediation is safer since it does not require excavation of contaminated soils. Also, it does not disturb the natural surroundings of the site,

Low cost compared to other remediation technologies.

Bioremediation has the following disadvantages [4]:

There is a potential for partial degradation to metabolites that are still toxic and/or potentially more highly mobile in the environment,

The process is highly sensitive to toxins and environmental conditions,

Extensive monitoring is required to determine biodegradation rates,

It may be difficult to control volatile organic compounds during ex-situ bioremediation process,

Generally requires longer treatment time as compared to other remediation technologies.

References [1] Anastas, P. T.; Warner, J. C., Green Chemistry: Theory and Practice, 1998, Oxford University Press Inc., New York. [2] Meagher, R.B. Current Opinion in Plant

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http://en.wikipedia.org/wiki/Bioremediation [5] http://www.genomenewsnetwork.org/categories/index/environment/toxic.php

Biology, 2000, 3 (2): 153-162. [3] Diaz E (editor), Microbial Biodegradation: Genomics and Molecular Biology, 1st ed., Caister Academic Press, 2008 [4] http://en.wikipedia.org/wiki/Bioremediation [5] http://www.genomenewsnetwork.org/categories/index/environment/toxic.php