Proposta di ricerca:
This proposal aims to investigate in the laboratory the potentiality of rhizo-assisted degradation of
polychlorinated biphenyls (PCBs) following a preliminary anaerobic degradation without plants.
The proposed activities will be carried out propaedeutically to the remediation of the PCB
contaminated site (SIN, DM Ambiente 8/7/02) of Papigno (Terni) as foreseen in a phytoremediation
plan submitted to the Environment Ministry and approved with few prescriptions. The industrial
site of Papigno was built around 1900 and closed around 1970, it was built on a surface of 105.450
m2 near the Nera River and Papigno village. The chemical plant produced calcium carbide
(CaC2) and Cianamide (CH2N2 / H2NCN). In front of the plant site there was an area used as a
landfill plant ("uncontrolled") for the industrial waste of the process. After the "industrial age", the
local municipality built two football fields and a little park on a portion of this area; the rest of the
area was abandoned. After the adoption of the national legislation for soil investigation the site was
characterized; the public park and the sport area were closed till the cleaning up of the area. The
main contaminanants of the soil and top soil are heavy hydrocarbons and PCB . The presence of
PCBs with the maximum concentration of 3 mg per Kg has been localized in two small spots of few
square metres and until 1 m depth. The approved plan is to remove all the PCB contaminated soil
and to place it in a confined area where it will be cleaned by applying plant assisted bioremediation.
PCBs are very difficult contaminants to remediate. They are a class of 209 synthetic congeners, in
which one to ten atoms are attached to a biphenyl ring (Ceccarini and Giannarelli, 2005). All
congeners are lipophilic, those with the highest number of chlorines are most recalcitrant because
chemically very stable, very toxic and. long range transportable. Their major source of
environmental contamination is the illegal or improper dumping of PCBs hazardous wastes. Italian
law imposes a concentration threshold limit of 1 microgram per Kg for industrial soils. Currently
used techniques for reducing PCB concentration, for example hydride reduction,
hydrodechlorination, dechlorination using metals, photolysis and gamma-radiation, oxidation,
electrolysis, mechano-chemical degradation are both of difficult application in situ and thus
expensive. Further, some of these methodologies may not degrade completely some PCB
congeners. For these reasons, and given the scarcity of economic resources in these years, the costeffective and ecofriendly technologies based on natural processes and proved to be efficient in
decontaminating soils from PCBs have gained more and more interest. Bioremediation is one of
these natural methods that is worth of consideration for degrading PCBs in soils (Ohtsubo et al.,
2004). Its limitation is that some PCBs, those with many chlorines, can be only partially degraded
by some bacteria under anaerobic conditions (Wegel and Wu, 2000). On the contrary, lower
molecular weight PCBs, included those originated from the anaerobic dechlorination, seem to be
completely degraded by other bacteria under aerobic conditions (Focht, 1995). Both processes,
aerobic and anaerobic, are essential to a rapid PCBs soil remediation and difficult that combine
under natural conditions. An opportunity is given in planted soil by rhizosphere activities where
apparently at microsite level the aerobic and anaerobic conditions would alternate every twenty
minutes due to spikes of oxygen release by plants to the rhizosphere soil (Smith, 1976). There are
no studies that apply these finding to the remediation of PCBs. This proposal within the bilateral
collaboration between CSIR and CNR, if funded, could be an opportunity to gain insights on these
rhizosphera phenomena and open to a further innovation of remediation technologies based on
natural methods. It has to be underlined, however, that not all plants can be applied for this
rhizoremediation studies. Among the few that could be very interesting are certainly salicaceae, a
family of trees that are very fast growing and can grow at very high density, up to 10,000 plants per
hectare (Marmiroli et al., 2011). These two characteristics seem to be favorable to the enhancement
of bacterial rhizoremediation under both aerobic and anaerobic conditions. It is, in fact, known that
fast and indeterminately growing plants need to fix high amount of carbon and nitrogen to sustain
more and more photosynthesizing and efficient organs. Nitrogen can be the limiting factor for this
investment and thus these plants need to send roots a lot of the photosynthetically fixed carbon
(between 20 and 30 percent) for increasing the root surface and then uptake more nitrogen from the
soil. The carbon sent to roots reaches apical cells where it should be used for root elongation
(Lynch, 1987). The high carbon concentration building up in these cells make root leaks of these
substances to the rhizosphere very likely. In this case all kinds of bacteria starving close to roots
will compete for this eventually leaked carbon. The simultaneous presence of some amount of
oxygen, also leaking from same cells in between the few mm distance from the root surface,
stimulated therein the proliferation and the activities of the aerobic microflora. The aerobic
condition lasts until oxygen concentration is depleted around creating de facto the condition for a
transient anaerobic condition. In synthesis, the rich and biodiverse microflora of the rhizosphere
could alternate their aerobic and anaerobic activity co-metabolizing even recalcitrant PCBs by
removing chlorines and preparing the biphenyl ring to the further or the complete aerobic breaking
down. The integration of these activities from a micro-rhizosphera level to a large rhizosphera such
that of a high density salicaceae plantation could be expected to effectively reduce the PCB soil
concentration of large contaminated sites. Such expectation can be will be tested by the two
collaborating groups that submit this proposal with the aim to study natural based methods to
rapidly destroy the recalcitrant forms of PCBs. At the same time the proposal will include research
activities focusing on the comprehension of various aspects of PCB biodegradation. For example,
the joint proposal will study 1) the role and the effectiveness of secondary plant metabolites in the
induction of specific metabolic degradation by specialized bacteria, 2) the effect of amendments
with biosurfactants on PCBs bioavailability.
Management of PCBs contaminated soils is thus going to be a big challenge in near future due their
persistence and toxicity to the environment. Innovative, cost-effective technologies based on natural
methods assisted by rhizosphere activities could be a formidable opportunity to cope with
environmental criticalities whose remediation is limited by the high economical costs.
Aon MA, Cabello M.N, Sarena D.E, Colaneri A.C, Franco M.G, Burgos J.L, Cortassa S 2000.
Spatio-temporal patterns of soil microbial and enzymatic activities in an agricultural soil. Applied
Soil Ecology 18(3 ) 239-254
Ceccarini, A and Giannarelli S 2005. Polychlorobiphenyls In Chromatographic Analysis of the
Environment, Third Edition. Nov 2005 , 667 -709
Focht, 1995. Strategies for improvement of aerobic metabolism of polychlorinated biphenyls. Curr
Opin Biotech 6:341-346
Lynch, J.M. 1987. The rhizosphere. Chichester: Wiley Interscience.
Marmiroli M, Pietrini F. Maestri E, Zacchini M, Marmiroli N and Massacci A (2011) Growth,
physiological and molecular traits in Salicaceae trees investigated for phytoremediation of heavy
metals and organics. Tree Physiol doi:10.1093/treephys/tpr088 (in press)
Ohtsubo Y, Kudo T., Tsuda M, Nagata Y. 2004. Strategies for bioremediation of polychlorinated
biphenyls. Appl. Microbiol Biot 65:250-258.
Smith A. M. 1976.Ethylene in soil biology. Ann Review Phytopatol 14:53-73.
Wegel J.and Wu Q. 2000. Microbial reductive dehalogenation of polychlorinated biphenyls. FEMS
Microbiology Ecology 32: 1-15.
Obiettivi:
The objectives of this study are 1) to screen candidate plants for assisting microbial PCB
degradation among fast growth trees that experimentally proved to release in the rhizosphere high
amount of carbon and oxygen; 2) characterization of rhizosphere and bulk soil microbial
populations for the ability to degrade PCB under aerobic and anaerobic conditions; 3) analysis of
soil amendments (micorrhizae to ehance plant growth, bio-surfactants to increase PCB mobility, cometabolites and metabolic inducers to activate the PCB degradation) to obtain higher rates of PCB
degradation under aerobic and anaerobic conditions; 5) bioaugmentation of microbial PCB
degraders under aerobic and anaerobic conditions and analysis of their persistence in the
contaminated soil; 6) microcosm tests with soil sampled from contaminated sites with PCB under
aerobic, anaerobic, xenic, and with or without the selected plants for rhizoremediation assisted PCB
degradation.
Pianificazione del lavoro
Piano di lavoro primo anno:
a) Sampling soil from contaminated area with 3,000 PCB micrograms per Kg for chemical
characterization of PCB congeners and for microcosms experiments.
b) Cutting harvesting from spontaneous salicaceae grown in the PCB contaminated area and their
set up test for three month growth under microcosms with contaminated soil in the glasshouse.
c) Analysis of the microbial community of the microcosms contaminated soil without plants and in
the rhizosphere soil with best growing plants by advanced biomolecular tools (fluorescence in situ
hybridization and quantitative PCR)
Piano di lavoro secondo anno:
a) Primary incubation for six months under anaerobic conditions of PCB contaminated soil
amended with i) ciclodextrins as biosurfactants for increasing PCB bioavailability, ii) biphenils for
inducing PCB metabolic degradation. Blanks will be carried out without both amendments, without
the biosurfactant and without the inducer.
b) Monthly chemical analysis by gas mass spectrometer of PCB congeners and degradation
metabolites on the incubated soil to assess the PCB degradation kinetic under anaerobic conditions
with the various amendments.
c) Aerobic incubation for further six months of the soil treated anaerobically eventually amended
with biosurfactants and/or inducers that resulted to increase significantly PCB degradation.
d) Parallel aerobic incubation as in c) with the addition of the selected plant cuttings to microcosms.
e) Monthly chemical analysis by gas mass spectrometer of PCB congeners and degradation
metabolites on the incubated soil to assess the PCB degradation kinetic under aerobic conditions
with the various amendments.
Piano di lavoro terzo anno:
a) Chemical analysis of soil with and without amendments and under anaerobic and under aerobic
conditions with and without plants and with and without amendments.
b) Statistical data analysis, kinetic elaboration, protocol writing for the experimental application in
the PCB contaminated site of Papigno of the set up decontamination methodology