Michel-Pierre Faucon
email: michel-pierre.faucon@lasalle-beauvais.fr

Soizig Le Stradic

Sylvain Boisson

Edouard Ilunga

Wa Ilunga

Maxime Séleck

Bastien Lange

Delhaye Guillaume

Mylor Ngoy Shutcha

Olivier Pourret

Pierre Meerts

Edouard Ilunga Wa Ilunga

Grégory Mahy

Implication of plant-soil relationships for conservation and restoration of copper-cobalt ecosystems

Keywords: biodiversity conservation, chemical soil factors, endemism, heavy metals, metallophyte, restoration ecology, soil-plant interactions

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

For decades, ecologists have attempted to understand the relationships between soil properties and plant diversity, as some soils are associated with high richness and endemism in plant species [START_REF] Whittaker | Scale and species richness: towards a general, hierarchical theory of species diversity[END_REF][START_REF] Escudero | Plant life on gypsum: a review of its multiple facets[END_REF]. A number of plant diversity hotspots are associated with nutrient-poor environments, notably in the Fynbos in South Africa, the Kwongan in southwestern Australia, and the Campos Rupestres in Brazil [START_REF] Cowling | Heterogeneity, speciation/extinction history and climate: explaining regional plant diversity patterns in the Cape Floristic Region[END_REF][START_REF] Hopper | The southwest Australian floristic region: evolution and conservation of a global hot spot of biodiversity[END_REF][START_REF] Lambers | Plant mineral nutrition in ancient landscapes: high plant species diversity on infertile soils is linked to functional diversity for nutritional strategies[END_REF][START_REF] Laliberté | How does pedogenesis drive plant diversity?[END_REF][START_REF] Hayes | Foliar nutrient concentrations and resorption efficiency in plants of contrasting nutrient-acquisition strategies along a 2-million-year dune chronosequence[END_REF][START_REF] Silveira | Ecology and evolution of plant diversity in the endangered campo rupestre: a neglected conservation priority[END_REF]. Regions with naturally metal-rich soils may also exhibit landscape and environmental heterogeneity, promoting high richness and endemism in plant species, such as New Caledonia [START_REF] Pillon | Ultramafic soils and species sorting in the flora of New Caledonia[END_REF], Sabah (Malaysia) (van der Ent et al. 2015a), California (USA) [START_REF] Brady | Evolutionary Ecology of Plant Adaptation To Serpentine Soils[END_REF], Cuba [START_REF] Borhidi | Phytogeography and vegetation ecology of Cuba[END_REF], and southeastern Democratic Republic of Congo (DRC) [START_REF] Küper | Africa's hotspots of biodiversity redefined[END_REF][START_REF] Faucon | Copper endemism in the Congolese flora: a database of copper affinity and conservational value of cuprophytes[END_REF]. The most widespread natural metalliferous outcrops across the world are ultramafic soils rich in nickel (Ni), chromium (Cr), iron (Fe), and magnesium (Mg) (Harrison and Rajakaruna 2011), but natural outcrops rich in Fe, manganese (Mn), copper (Cu), cobalt (Co), and other rare minerals also occur. These metalliferous outcrops support unique vegetation adapted to high metal concentrations (i.e., Ni, Cr, Fe, or Mg) and, in general, nutrient deficiency and cation imbalances. Vegetation occurring in metal-rich habitats may present an important proportion of endemic species [START_REF] Rajakaruna | The Edaphic Factor in the Origin of Plant Species[END_REF][START_REF] Faucon | Copper endemism in the Congolese flora: a database of copper affinity and conservational value of cuprophytes[END_REF]Arnacker 2011). These endemics significantly contribute to the biodiversity of some regions of the world [START_REF] Kruckeberg | Biological Aspects of Endemism in Higher Plants[END_REF]Jacobi et al. 2007;[START_REF] Anacker | Phylogenetic conservatism in plantsoil feedback and its implications for plant abundance[END_REF]van der Ent et al. 2015b). For example, in California, endemic species from ultramafic rock outcrops represent close to 12% of the state endemic flora [START_REF] Kruckeberg | Endemic metallophytes: their taxonomic, genetic and evolutionary attributes[END_REF][START_REF] Safford | Serpentine endemism in the California flora: a database of serpentine affinity[END_REF]; in New Caledonia, they correspond to almost 60% of the island's indigenous flora (Jaffré 1992).

In addition, metallicolous flora represents a remarkable biological resource for eco-technological applications, especially phytoremediation of contaminated soils (Boisson et al. In press;[START_REF] Shutcha | Phytostabilisation of copper-contaminated soil in South-East DRC: an experiment with three native grasses and two amendments[END_REF][START_REF] Shutcha | Three years of phytostabilisation experiment of bare acidic soil extremely contaminated by copper smelting using plant biodiversity of metal-rich soils in tropical Africa (South-East DRC, DR Congo)[END_REF]van der Ent et al. 2015c). [START_REF] Whiting | Research Priorities for Conservation of Metallophyte Biodiversity and their Potential for Restoration and Site Remediation[END_REF] considered these species an "El Dorado" of genetic material, which could be used for decontamination or ecological restoration of metal-contaminated sites.

Implications in eco-technologies are particularly enhanced with the valorisation of metals in biomass from hyper-accumulators in green chemistry (Zn, Ni, Platinium -Pt, Cu, Co, and Manganese -Mn) as catalysts in organic synthesis of molecules of interest [START_REF] Losfeld | The chemical exploitation of nickel phytoextraction: an environmental, ecologic and economic opportunity for New Caledonia[END_REF][START_REF] Escande | From biodiversity to catalytic diversity: how to control the reaction mechanism by the nature of metallophytes[END_REF][START_REF] Grison | Combining phytoextraction and ecocatalysis: a novel concept for greener chemistry, an opportunity for remediation[END_REF].

As metallicolous vegetation often occurs on economically valuable mineral deposits, many are threatened by quarrying and mining activities [START_REF] Erskine | Sustaining metal-loving plants in mining regions[END_REF][START_REF] Faucon | May Rare Metallophytes Benefit from Disturbed Soils Following Mining Activity? The Case of the Crepidorhopalon tenuis in South-East DRC (D. R. Congo)[END_REF][START_REF] Whiting | Research Priorities for Conservation of Metallophyte Biodiversity and their Potential for Restoration and Site Remediation[END_REF]. Moreover, extraction processes may lead to the contamination of adjacent nonmetalliferous habitats by industrial wastes (smelts, waste water, etc.), which might impact local plant diversity. Establishment of conservation and/or restoration programmes for plant biodiversity of metal-rich habitats (in-situ and ex-situ conservation) requires the understanding of relationships between soil and plant diversity. Knowledge of ecological and biogeochemical processes governing ecosystems, communities, and populations is fundamental to defining and applying ecological restoration of disturbed habitats (Palmer et al. 1997).

In southeastern DRC, natural copper and cobalt outcrops (Cu-Co outcrops) host remarkable herbaceous communities that comprise the so-called "copper flora". These outcrops form isolated and scattered hills in a landscape matrix of Miombo woodland [START_REF] Duvigneaud | Cuivre et végétation au South-East DRC[END_REF][START_REF] Duvigneaud | Plantes cobaltophytes dans le Haut South-East DRC[END_REF]Fig. 1) (http://copperflora.org/eflora/). The Cu-Co outcrops present a variety of habitats according to a variation of edaphic conditions, including the natural Cu-Co contamination level of the soil (Fig. 1). Mineralisation of the parent rocks and geological succession promotes the Cu and Co enrichment of the soils along the slope of the hills with concentrations of bioavailable Cu from 20 to 10,000 mg.kg -1 and Co from 2 to 1,000 mg.kg -1 toward the top of the Cu hills. These plant communities host more than 600 species tolerant to high Co and Cu concentrations [START_REF] Leteinturier | Evaluation du potential phytocénotique des gisements cuprifères d'Afrique centro-australe en vue de la phytoremédiation de sites pollués par l'activité minière[END_REF]. Among those tolerant species, 57 have been identified as endemic from the Cu-Co outcrops (i.e., Cu-Co endemics). The region, due to its extremely metal-rich subsoil, is currently at the forefront of mining activity. In DRC, 70% of metallophytes taxa (species or genera) are considered critically endangered (i.e., CR) and about 10% would already have disappeared [START_REF] Faucon | Copper endemism in the Congolese flora: a database of copper affinity and conservational value of cuprophytes[END_REF]2012a). In addition, southeastern DRC is one of the principal regions across the world presenting both environmental and public health issues associated with soil, air, and water contaminations resulting from an important Cu, Co, and uranium (U) extraction [START_REF] Banza | High human exposure to cobalt and other metals in South-East DRC, a mining area of the Democratic Republic of Congo[END_REF][START_REF] Manda | Evaluation de la contamination de la chaîne trophique par les éléments traces (Cu, Co[END_REF][START_REF] Cheyns | Pathways of human exposure to cobalt in South-East DRC, a mining area of the DR Congo[END_REF]. There is an urgent need to develop conservation measures as well as restoration projects for the biodiversity of Cu and Co outcrops. This paper reviews recent advances in our understanding of relationships between soil and plant diversity in metalliferous outcrops located in southeastern DRC and the implication of these recent advances in defining conservation and restoration strategies for the Cu-Co ecosystems and their associated plant biodiversity.

Patterns of Species Richness and Endemism on Copper-cobalt Outcrops

High Cu and Co concentrations in soil are phytotoxic and represent a strong selection pressure for plant species, which may induce ecological isolation and promote the speciation process [START_REF] Duvigneaud | Cuivre et végétation au South-East DRC[END_REF][START_REF] Brooks | Metal-enriched sites in south central africa[END_REF][START_REF] Macnair | The Evolution of Edaphic Endemics[END_REF]. Among the approximate 600 plant species of flora of Cu-Co outcrops, 32 are strictly Cu-Co endemic (i.e., absolute metallophyte occurring exclusively on Cu-Co rich soils), and 23 are broad Cu-Co endemics (i.e., facultative metallophyte with more than 75% of known populations occurring on Cu-Co rich soils) [START_REF] Faucon | Copper endemism in the Congolese flora: a database of copper affinity and conservational value of cuprophytes[END_REF]. At outcrop scale, Cu and Co concentrations are a primary determinant of the richness of plant species. Outcrops with the highest Cu and Co concentrations in soil support the lowest total plant species richness [START_REF] Duvigneaud | Cuivre et végétation au South-East DRC[END_REF][START_REF] Saad | Investigating the Vegetation-Soil Relationships on the Copper-Cobalt Rock Outcrops of South-East DRC (D. R. Congo), an Essential Step in a Biodiversity Conservation Plan[END_REF][START_REF] Séleck | Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration[END_REF]). This pattern is in contrast to the richness of endemic metallophyte, which rises with the increase of Cu and Co concentration in the soil [START_REF] Saad | Investigating the Vegetation-Soil Relationships on the Copper-Cobalt Rock Outcrops of South-East DRC (D. R. Congo), an Essential Step in a Biodiversity Conservation Plan[END_REF][START_REF] Séleck | Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration[END_REF]. At the landscape scale, the spatial configuration of Cu-Co outcrops influences the richness of Cu-Co endemics of species. In a recent study of 34 Cu-Co outcrops varying in size (0.2 ha to 27.3 ha) and geographical isolation in a 30*20 km landscape, Ilunga wa Ilunga et al. (unpublished results) demonstrated that Cu-Co endemic richness is positively correlated to the site surface, which might be associated with higher habitat diversity. Using species accumulation curves, it was demonstrated that the three largest outcrops encapsulate the total Cu-Co endemic richness (25 taxa) of the landscape, whereas at least 15 small Cu-Co outcrops are necessary to reach the same endemic richness.

How does endemic metallophyte richness in southeastern DRC compare with other metallicolous floras? The proportion of strict metallophyte endemic in the copper flora, estimated at 5%, is obviously low when compared with ultramafic floras of California, Cuba, and New Zealand but is rather similar to ultramafic floras of Italy or Great Dyke (Fig. 2). Endemic richness and the endemism percentage depend on a combination of different variables [START_REF] Harrison | Regional and local species richness in an insular environment: serpentine plants in California[END_REF]. The history of geographical isolation is important to explain endemism patterns.

Cuba and New Caledonia are oceanic islands with a long history of geographical isolation resulting in a high global level of endemism (Fig. 2). The age of exposure of metalliferous outcrops may also be an important factor to explain the proportion of endemics in a metallicolous flora [START_REF] Harrison | Does the Age of Exposure of Serpentine Explain Variation in Endemic Plant Diversity in California?[END_REF]. Copper mineralisation in southeastern DRC dates from the late Cambrian period (about 620 myr, François 1973)), but Cu-Co rich rocks have been exposed to plant colonisation for a much shorter period. Recent data indicate 2-3 myr as a likely age for Cu-Co outcrops [START_REF] Putter | Malachite, an indicator of major Pliocene Cu remobilization in a karstic environment (South-East DRC, Democratic Republic of Congo)[END_REF]. This is similar to the age of exposure of California ultramafic outcrops [START_REF] Harrison | Does the Age of Exposure of Serpentine Explain Variation in Endemic Plant Diversity in California?[END_REF]. Vegetation modification due to palaeoclimate variation might also be relevant to explain the endemism level in metallicolous floras. During the Holocene, tropical Africa experienced dramatic climate fluctuations, including a dry-cool period about 18,000 cal yr BP [START_REF] Van Zinderen Bakker | A review of late quaternary pollen studies in East, Central and Southern Africa[END_REF][START_REF] Vincens | A 23,000 yr pollen record from Lake Rukwa (8°S, SW Tanzania): New data on vegetation dynamics and climate in Central Eastern Africa[END_REF], with a deglacial period between 16,500 and 12,300 cal yr BP, including a retreat of montane elements at higher altitudes on the plateau under warmer conditions. This led to evolutionary divergences between populations isolated on the Cu-Co outcrops. These relatively recent divergences might explain the low percentage of endemic species observed in the copper flora. The close morphological resemblance between Cu-Co endemic plant species and more widespread counterparts, such as Vigna dolomitica and V. reticulata [START_REF] Maxted | Palatability of Thlaspi caerulescens for snails: influence of zinc and glucosinolates[END_REF]), Crotalaria peschiana and C. subcaespitosa [START_REF] Polhill | Crotalaria in Africa and Madagascar[END_REF], Acalypha cupricola, A. fuscescens, and A. dikuluwensis [START_REF] Levin | Two New Species of <I>Acalypha</I> (Euphorbiaceae) from Tropical Africa, and a Review of Some Robyns Names for Cupricolous Plants From the Democratic Republic of the Congo[END_REF], and Silene burchellii and S. cobalticola [START_REF] Malaisse | Phytogeography of the copper and cobalt flora of Upper Shaba (Zaïre), with emphasis on its endemism, origin and evolution mechanisms[END_REF], suggests that the speciation processes may be ongoing in the southeastern DRC region. This also supports the hypothesis that most of Cu-Co endemic species are neo-endemics [START_REF] Malaisse | Phytogeography of the copper and cobalt flora of Upper Shaba (Zaïre), with emphasis on its endemism, origin and evolution mechanisms[END_REF][START_REF] Brooks | The Heavy Metal Tolerant Flora of Southcentral Africa: a mutlidisciplinary approach[END_REF][START_REF] Wu | Colonisation and establishment of plants in contaminated sites[END_REF]. This implies a recent divergence under intense ecological isolation [START_REF] Macnair | The Evolution of Edaphic Endemics[END_REF][START_REF] Rajakaruna | The Edaphic Factor in the Origin of Plant Species[END_REF]). Another factor that may contribute to the low metallophyte endemism in southeastern DRC is the relatively low total surface of mineralised soils. Based on a typical site surface of a few tenths of a hectare, the total area of the Cu-Co outcrops may not exceed 100 km², which is a small surface compared to the thousands of km² of ultramafic soil existing in California, Cuba, or New Caledonia (Harrison and Rajakaruna 2011). Eventually, ecological isolation and selective forces acting on populations occurring on Cu-Co rich soils may be overestimated. Unlike ultramafic soils, Cu-Co rich soils of southeastern DRC are relatively rich in nutrients (P, Ca, and Mg) [START_REF] Faucon | May Rare Metallophytes Benefit from Disturbed Soils Following Mining Activity? The Case of the Crepidorhopalon tenuis in South-East DRC (D. R. Congo)[END_REF][START_REF] Séleck | Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration[END_REF], and Cu soil toxicity might be mitigated by organic matter and other metals in oxidated forms [START_REF] Lange | Prediction of the edaphic factors influence upon the copper and cobalt accumulation in two metallophytes using copper and cobalt speciation in soils[END_REF][START_REF] Pourret | Modeling of cobalt and copper speciation in metalliferous soils from South-East DRC (Democratic Republic of Congo)[END_REF].

Even if metal-rich habitats are favourable environments for evolutionary divergence, the exceptionally high endemism occurring in such habitats remains intriguing [START_REF] Kay | Plant speciation. Serpentine: the evolution and ecology of a model system[END_REF]. Intrinsic characteristics of metaltolerant taxa may be responsible for the limitation of their ecological niche. One hypothesis for high endemism in metal-rich habitats might be a low ability of metallophyte to colonise non-metalliferous habitats due to constitutive needs in metals [START_REF] Tadros | Evidence of the presence of an edapho-biotic factor in the problem of serpentine tolerance[END_REF][START_REF] Kay | Plant speciation. Serpentine: the evolution and ecology of a model system[END_REF]. For some Cu-Co endemic taxa, it has been demonstrated that biomass and fitness increase with Cu concentration in soil [START_REF] Chipeng | Copper tolerance in the cuprophyte Haumaniastrum katangense (S. Moore) P.A. Duvign. & Plancke[END_REF]. Metal tolerance may also represent an adaptive cost responsible for a disadvantage of metallophyte in nonmetalliferous soils [START_REF] Wu | Colonisation and establishment of plants in contaminated sites[END_REF]Macnair et al. 2000) with a reduced competitive ability of metallophyte in nonmetalliferous habitats. Some studies support the hypothesis that the restricted distribution of metallophyte on metal-rich soils could be due to their low resistance to pathogens. Metal toxicity constitutes a strong selection pressure against pathogens and herbivores, limiting pathogen and herbivory pressures on metallophytes (Noret et al. 2005). As a result, metal tolerant taxa may also be characterised by a decrease in defence against herbivory and pathogens, which could limit their colonisation of non-metalliferous habitats (Faucon et al. 2012b;[START_REF] Kazakou | Hypotheses, mechanisms and trade-offs of tolerance and adaptation to serpentine soils: From species to ecosystem level[END_REF]. Very few data exist for Cu flora, but it has been shown that the Cu-Co endemic Crepidorhopalon perennis is able to grow on substrate without Cu only in axenic conditions (i.e., without soil biota) (Faucon et al. 2012b). However, this hypothesis is still controversial because some species of bacteria and fungi are perfectly adapted to metal-rich soils [START_REF] Wakelin | Mechanisms of pollution induced community tolerance in a soil microbial community exposed to Cu[END_REF][START_REF] Stefanowicz | Metals affect soil bacterial and fungal functional diversity differently[END_REF].

As a result of the complex interactions among physiological, ecological, and evolutionary factors, the ecological niches of Cu-Co endemic from southeastern DRC vary widely [START_REF] Faucon | May Rare Metallophytes Benefit from Disturbed Soils Following Mining Activity? The Case of the Crepidorhopalon tenuis in South-East DRC (D. R. Congo)[END_REF]2012a). Congeneric species may present highly distinct edaphic niches. Crepidorhopalon perennis (P.A. Duvigneaud) Eb. Fisch.

(Linderniaceae), a Cu-Co endemic, occurs on soils richer in Cu compared to its pseudo-metallophyte congener C. tenuis (S. Moore) Eb. Fisch. [START_REF] Faucon | May Rare Metallophytes Benefit from Disturbed Soils Following Mining Activity? The Case of the Crepidorhopalon tenuis in South-East DRC (D. R. Congo)[END_REF]2012b)

Soil and Plant Communities' Co-variation.

The physiognomy of vegetation varies on Cu-Co outcrops from the top to the bottom along a topographical gradient corresponding roughly to the Cu/Co gradient (Fig. 1). At the top, chasmophytic vegetation generally develops on poorly mineralised rocks (i.e., plant communities colonising the cracks and fissures of low mineralised rock with Cu concentrations of 250-900 mg kg -1 ). Steppe vegetation colonises the upper part of the outcrops with the highest Cu soil concentrations (ranging from 3,500 to 10,000 mg kg -1 ). Finally, steppic savannah vegetation develops on the intermediate and foothill slopes and flat periodically flooded savannahs (dembos) at the bottom of the outcrops with Cu concentrations varying from 100 to 3,500 mg kg -1 [START_REF] Duvigneaud | Cuivre et végétation au South-East DRC[END_REF][START_REF] Brooks | The Heavy Metal Tolerant Flora of Southcentral Africa: a mutlidisciplinary approach[END_REF][START_REF] Saad | Investigating the Vegetation-Soil Relationships on the Copper-Cobalt Rock Outcrops of South-East DRC (D. R. Congo), an Essential Step in a Biodiversity Conservation Plan[END_REF][START_REF] Séleck | Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration[END_REF]) (Fig. 1). However, vegetation proved to be more complex than a simple succession of physiognomic plant formations along correlated Cu and Co gradients. More detailed studies revealed a mosaic of plant communities differing in species assemblages within and among Cu-Co outcrops [START_REF] Saad | Investigating the Vegetation-Soil Relationships on the Copper-Cobalt Rock Outcrops of South-East DRC (D. R. Congo), an Essential Step in a Biodiversity Conservation Plan[END_REF][START_REF] Séleck | Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration[END_REF][START_REF] Wa Ilunga | Small-scale diversity of plant communities and distribution of species niches on a copper rock outcrop in Upper South-East DRC, DR Congo[END_REF].

Although (extractable) Cu and Co soil concentrations are the main factors correlated to variation in species composition among plant communities on Cu-Co outcrops, the mosaic of plant communities is also related to complex variations of other inter-correlated chemical factors. For example, in addition to differences in Cu-Co concentrations, steppic communities present higher values for pH, C, N, and extractable Ca and P (acetate-EDTA) and lower values for the C:N ratio and extractable Fe and K concentrations compared to steppic savannah communities. Besides the potential direct effect on species assemblages, soil factors, such as pH, C, Ca and Mn, can also contribute indirectly to floristic variation and heterogeneity of plant communities in Cu outcrops [START_REF] Saad | Investigating the Vegetation-Soil Relationships on the Copper-Cobalt Rock Outcrops of South-East DRC (D. R. Congo), an Essential Step in a Biodiversity Conservation Plan[END_REF][START_REF] Wa Ilunga | Small-scale diversity of plant communities and distribution of species niches on a copper rock outcrop in Upper South-East DRC, DR Congo[END_REF][START_REF] Séleck | Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration[END_REF] because they can influence Cu and Co availability and toxicity [START_REF] Lange | Prediction of the edaphic factors influence upon the copper and cobalt accumulation in two metallophytes using copper and cobalt speciation in soils[END_REF][START_REF] Pourret | Modeling of cobalt and copper speciation in metalliferous soils from South-East DRC (Democratic Republic of Congo)[END_REF]. Cobalt has a high affinity for manganese oxides (MnOx). A higher MnOx concentration in soil can decrease Co availability and toxicity [START_REF] Collins | Pedogenic factors and measurements of the plant uptake of cobalt[END_REF]). In the same way, high concentrations of iron oxides and organic matter in soils can reduce Cu availability [START_REF] Kabala | Fractionation and mobility of copper, lead, and zinc in soil profiles in the vicinity of a copper smelter[END_REF]. Characterisation of metal speciation in soils also suggests a strong relationship between Cu and Co speciation in soils and structures of plant communities on a single Cu-Co outcrop (Fig. 3). A steppe community (Community 3 in Fig. 3) is associated with high concentrations of Cu and Co fractions that are considered available (i.e., Cu-Free, Cu-FeOx (Cu-iron oxides), Cu-MnOx, Co-Free, and Co-FeOx) [START_REF] Lange | Prediction of the edaphic factors influence upon the copper and cobalt accumulation in two metallophytes using copper and cobalt speciation in soils[END_REF]. A second steppe community (Community 2 in Fig. 3) presents the highest concentrations of unavailable Cu and Co fractions (i.e., Cu-OM and Co-MnOx) and less Cu-Co-tolerant species. Variation of Cu and Co chemical forms in soils may create spatial heterogeneity of soil properties that promotes a diversity of plant assemblages (Fig. 4). It may be hypothesised that the diversity of Cu and Co chemical forms in soil decreases metal availability and toxicity and promotes plant species diversity.

Nutrient content does not seem restrictive for the vegetation of Cu-Co outcrops; the amount of the essential macronutrients is higher on Cu-Co outcrops than on non-metalliferous soils in southeastern DRC [START_REF] Saad | Investigating the Vegetation-Soil Relationships on the Copper-Cobalt Rock Outcrops of South-East DRC (D. R. Congo), an Essential Step in a Biodiversity Conservation Plan[END_REF][START_REF] Séleck | Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration[END_REF]. This differs from ultramafic soils where deficiencies in N, P, K, and Ca have been suggested as a potential reason for limited plant productivity [START_REF] O'dell | Congeneric serpentine and nonserpentine shrubs differ more in leaf Ca:Mg than in tolerance of low N, low P, or heavy metals[END_REF][START_REF] Whittaker | The Ecology of Serpentine Soils[END_REF]. It can be thus inferred that selection pressure may be more strongly influenced by metal toxicity than by a high variation of nutrient content. However, the covariation of plant communities with nutrients, partly independent from metal availability (Seleck et al. 2013), suggests the need for a deeper exploration of the effect of N and P through the examination of the variation of the foliar N:P ratio and experimentation on P limitations along a Cu/Co gradient, where both the diversity and productivity of plant species should be measured [START_REF] Aerts | The mineral nutrition of wild plants revisited: a re-evaluation of processes and patterns[END_REF].

Variations of Cu and Co concentrations in soil also involve variations in trait responses of plant species. Delhaye et al. (unpublished results) showed that the gradient of soil metal concentrations is associated with a pattern of trait substitution rather than high intraspecific trait variation. At the community-level, trait variation results in a shift in the abundances of various life forms. Xylopod species (i.e., with underground storage organs) are dominant in the lower part of the Cu-Co outcrops in communities occurring on deeper soils that are less rich in metal, whereas annual species are dominant in the upper part of the Cu-Co outcrop in communities located on more shallow soils with high metal concentrations [START_REF] Séleck | Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration[END_REF].

Implication of Plant-soil Relationships for Conservation and Restoration of Plant Biodiversity in Cu-Co

Outcrops in Southeastern DRC

The first strategy from a biodiversity conservation point of view would include the preservation of a proportion of Co-Cu outcrops in protected areas. As demonstrated by endemism patterns at the landscape scale, the preservation of a limited number of Cu-Co outcrops occurring in southeastern DRC should allow the conservation of a set of populations of most Cu-Co endemic species. In contrast, the preservation of the diversity of plant communities in untouched protected Cu-Co outcrops will be far more challenging. The high diversity of plant communities as well as the variability of plant assemblages among outcrops would need to preserve a large proportion of Cu-Co outcrops if ecosystem diversity must be addressed. This may turn out to be a limited and economically unrealistic option, considering the increasing demand for Cu and Co in the world and the high contribution of the Cu-Co market to the economy of the DRC.

A valuable alternative to preserving the extraordinary biological heritage of Cu-Co outcrops is to set up, prior to ecological restoration and rehabilitation of sites at mine closure, an ex-situ conservation strategy at the level of plant communities with topsoil and community translocation and at the level of individual species with translocation of individuals in restored habitats, conservation and multiplication in botanical gardens, and long term ex-situ seed banks for most characteristic species (http: //www.copperflora. org: Conservation; Godefroid et al. 2013) (Fig. 5).

In this review, we have highlighted that extractable Cu-Co soil concentrations and other edaphic soil factors (i.e., pH, C, N, Ca, Mn, and Fe) are the main drivers structuring plant communities, and their variation is partly responsible for the heterogeneity of communities observed at the scale of the Cu-Co outcrops. Alteration and/or modifications of edaphic conditions of Cu-Co outcrops therefore have important consequences on vegetation composition and structure. Plant communities of Cu-Co outcrops are poorly or not resilient to strong anthropogenic disturbances, such as mineral extraction, made either by artisanal miners or mining companies (Ilunga wa [START_REF] Wa Ilunga | Plant functional traits as a promising approach for the ecological restoration of degraded tropical metal-rich habitats and revegetation of metal-rich bare soils[END_REF]. The comparison, in terms of species composition and/or abundance of functional traits, between primary plant communities occurring on Cu-Co outcrops and secondary plant communities resulting from the re-colonisation of disturbed areas (i.e., altered areas after mineral extraction with potentially deposition of mining waste) did not show any functional resilience of the primary communities, even after 30 years of degradation by mining [START_REF] Faucon | May Rare Metallophytes Benefit from Disturbed Soils Following Mining Activity? The Case of the Crepidorhopalon tenuis in South-East DRC (D. R. Congo)[END_REF][START_REF] Wa Ilunga | Plant functional traits as a promising approach for the ecological restoration of degraded tropical metal-rich habitats and revegetation of metal-rich bare soils[END_REF]. Conservation or restoration of the soil factors is therefore necessary and crucial to conserve plant community on Cu-Co outcrops.

Plant community translocation projects through the transfer of topsoil and vegetation mats have been set up in southeastern DRC by Tenke Fungurume Mining, a mining company (Fig. 5). The initial results of these translocations demonstrate that vegetation mat translocation is the most efficient method to preserve the biodiversity of Cu-Co outcrops compared to topsoil transfer (Le Stradic et al. In press). In contrast to topsoil transfer, vegetation mat translocation allows transference of numerous species with fewer non-target species, probably due to a higher competition with the already established vegetation. For steppic savannahs, the seed bank is poor in species and seeds, and little emergence of target species (i.e., copper flora species) occurs in the topsoil, while ruderal species quickly colonise bare ground areas. Results are more encouraging for the steppe, given that high metal concentrations (i.e., Cu and Co) appear to limit the development of ruderal species, and a greater number of annual species provides a more rapid vegetation cover from the first year with some target species of copper flora (i.e., species present in the pristine Cu-Co communities), such as Bulbostylis cupricola Ex-situ conservation of individual species also introduces great challenges. In this review, we have highlighted interspecific variations in realised ecological niches of Cu-Co endemics in relation to Cu and Co concentrations in soils but also other chemical and physical soil factors. This suggests that conservation strategies need to be species-specific and cannot be generalisable for all endemic metallophytes, particularly if the aim of the ex-situ conservation strategy is reintroducing and self-sustaining populations in restored habitats. In the short term, detailed studies are needed to characterise both the fundamental and realised niches of endemic metallophytes from Cu-Co outcrops [START_REF] Schenk | Soil depth, plant rooting strategies and species' niches[END_REF]. Characterisation of the fundamental niches of Cu-Co endemics (i.e., the physiological tolerance of a species in the absence of biotic interactions) will be crucial to developing ex-situ conservation and multiplication programmes in botanical gardens before reintroduction in restored habitats.

Although species conservation priorities are generally based on rarity and species extinction threats (IUCN criteria), conservation strategies for Cu-Co metallophytes should also integrate the conservation of genetic diversity within the region. Some genetic variation of adaptive traits exists between populations of the same species. Genetic variation between populations of Cu tolerance and accumulation has been demonstrated in two pseudo-metallophytes, Crepidorhopalon tenuis and Haumaniastrum katangense (Faucon et al. 2012b;[START_REF] Peng | Differences in copper accumulation and copper stress between eight populations of Haumaniastrum katangense[END_REF]. This genetic variability of Cu tolerance and accumulation is an opportunity to select the most tolerant populations and define phytoremediation processes (phytoextraction or phytostabilisation).

Conclusion

This review highlights advances in knowledge of plant diversity of natural Cu-Co outcrops at different scales or ecological levels (flora, ecosystems, plant species communities, and plant populations) in order to provide guidelines and identify knowledge gaps to define biodiversity conservation programmes. In mining regions where there is an obvious conflict of interest between economically important mining activities and conservation of plant biodiversity of natural metalliferous outcrops, there is an urgent need to define science-based strategies for biodiversity conservation, including in-situ and ex-situ approaches, ecosystem reconstruction, and postmining restoration (Fig 5). Ex-situ conservation prior to mining activities could be undertaken on available areas adjacent to mineralised outcrops with Cu-Co rich soils or Cu-Co enriched soils. Plant diversity, especially metallophytes, even in ex-situ conservation areas, is a genuine resource for phytoremediation of degraded postmining areas. Future challenges are to conciliate biodiversity conservation and ecological engineering for phytoremediation of Cu-Co contaminated soils generated by mining activities. This goal will be reached only if a strong cooperation between scientists, field conservationists, and mining companies is set up. Considering the urgency of the situation, scientific studies should be a full part of conservation strategies. In turn, learning from true conservation experience needs to be designed now (research by design) and will be a key method for increasing our scientific knowledge of this exceptional biological resource.

  . Boisson et al. (unpublished results) recently demonstrated that among eight Cu-Co endemic species present on the same Cu-Co outcrops, five have their optimum niches in the lowest concentrations of Cu (< 300 mg Cu.kg -1 ) and Co (55 mg Co.kg -1 ) and only two have their optimum niches in the highest concentrations of Cu (> 5,000 mg Cu.kg -1 ). Species with their optimums in the higher Cu/Co concentrations also present the largest Cu/Co niche width. Ilunga wa Ilunga et al. (2013) found a similar pattern for non-endemic tolerant species on a Cu-Co outcrop in southeastern DRC. In addition, Ilunga wa Ilunga et al. (2013) demonstrated niche differentiation in relation to physical soil characteristics, namely rock cover and percentage of stones in the soil.

  Goetgh. or Haumaniastrum robertii (Robyns) P.A. Duvign. and Plancke. Steppic communities present shallow soils, favouring the transfer of the plant community without root damage. In contrast, vegetation mat translocation failed to transfer structuring xylopod species (i.e., dominant species) with important underground systems, such as Cryptosepalum maraviense (Fabaceae). The absence of xylopods in translocated ecosystems may modify underground competition relationships within the community and promote the dominance of Poaceae species. While edaphic conditions are essential in order to restore Cu-Co communities properly, biotic filters and species interactions are also necessary to structure plant communities occurring on Cu-Co outcrops, and more research on this topic is necessary.

Figures 518 Fig. 1

 5181 Figures 518

Fig. 2

 2 Fig.2Relationship between total plant species richness and metallophyte endemic richness on natural metal rich soils in six metal world regions. Data are from[START_REF] Faucon | Copper endemism in the Congolese flora: a database of copper affinity and conservational value of cuprophytes[END_REF] Wild 1965; Jaffré 1992;[START_REF] Borhidi | Phytogeography and vegetation ecology of Cuba[END_REF] Kruckeberg 1984; Selvi 2007.

Fig. 3

 3 Fig. 3 NonMetrical Multidimensional scaling (NMDS) ordination diagram of plots (n=83) distributed in three different communities on a Cu-Co outcrop called Fungurume V. Fitted copper and cobalt fractions vectors are overlain by using the envfit command of vegan package (R statistical software, Oksanen et al. 2011). MnOx: manganese oxides. FeOx: iron oxides. Community 1: Steppic savannah with Cryptosepalum maraviense (Caesalpiniaceae); Loudetia simplex (Poaceae) et Scleria bulbifera (Cyperaceae) (Figure 1 photo g and h). Community 2: Steppe with Hyparrhenia diplandra (Poaceae); Schizachyrium brevifolium (Poaceae); Justicia elegantula (Acanthaceae), Michrochloa altera (Poaceae); Xerophyta equisetoides (Velloziaceae) and Bulbostylis cupricola (Cyperaceae) (Figure 1 photo f). Community 3: Steppe with Pandiaka carsonii (Amaranthaceae);Ascolepis metallorum (Cyperaceae); Anisopappus davyi (Asteraceae) (Fig.1).

Fig. 4

 4 Fig. 4 Relationship between 1/D Simpson diversity (D = Simpson's dominance index) of copper chemical forms in soils and Cu Shannon diversity of higher plant species (n=83). Copper fractions in soils were modeled by speciation modeling (WHAM 6) from method of Pourret et al. 2015.

Acknowledgments

The authors are grateful to Hans Lambers and Etienne Laliberté (School of Plant Biology, University of Western Australia) and Antony van der Ent (Centre for Mined Land Rehabilitation, University of Queensland) for relevant comments and language revision of the manuscript. University of Lubumbashi and the NGO Biodiversité au southeast DRC (BAK) are gratefully acknowledged for the welcome given to MPF. We thank the company Tenke Fungurume Mining (TFM) for the logistic and financial support to conduct part of our study and the Fonds de la Recherche dans l'Industrie et dans l'Agriculture (FRIA) of the FNRS (Fonds National de la Recherche Scientifique), Belgium for their financial support.


Bersillon

V Kazpard

B S Lartiges
email: bruno.lartiges@get.omp.eu

C Frochot

J B D'espinose De La Caillerie

M L Viriot

J M Portal

T Görner

J L Bersillon

FATE OF COAGULANT SPECIES AND CONFORMATIONAL EFFECTS DURING THE AGGREGATION OF A MODEL OF A HUMIC SUBSTANCE WITH AL 13 POLYCATIONS

Keywords: Coagulation, humic substances, Al 13 polycation

Fate of coagulant species and conformational effects during the aggregation of a model of a humic substance with Al13 polycations

INTRODUCTION

Optimizing natural organic matter (NOM) removal by coagulation is a crucial challenge in drinking water treatment. Indeed, the organic material remaining within the clarified water is involved in most problems encountered in the treatment process such as reduced adsorption capacity in activated carbon beds, formation of carcinogenic chlorinated organic compounds during disinfection, taste and odour of the finished water, and biological regrowth in the distribution networks [START_REF] Owen | NOM characterization and treatability[END_REF]. Several mechanisms have been proposed to explain the interaction of NOM with hydrolyzed coagulant species. Charge neutralization is thought to occur at low pH as suggested by stoichiometric relationships between coagulant demand and dissolved organic matter concentration, or restabilization upon overdosing (Narkis and Rehbun, 1977), whereas under conditions favoring metal hydroxide precipitation, physical entrapment and/or adsorption of organic matter onto the freshly formed precipitate are assumed to play a major role in NOM removal [START_REF] Bose | Adsorption of natural organic matter on preformed aluminum hydroxide flocs[END_REF].

However, none of the above destabilization mechanisms account for the low removal efficiency which does not exceed 60 percent in most cases [START_REF] Jacangelo | Selected processes for removing NOM[END_REF]. NOM is in fact a very complex mixture of organic components derived from soils or produced within surface waters and sediments by biological and chemical processes. Recent research has shown that larger molecular weight molecules are more effectively removed than smaller ones, and that the hydrophobic fraction of NOM is more easily destabilized by coagulation than hydrophilic compounds [START_REF] Collins | Molecular weight distribution, carboxilic acidity, and humic substances content of aquatic organic matter : implications for removal during water treatment[END_REF]Bose and Reckow, 1998). Selectivity also depends on coagulant nature, as polysaccharides are more amenable to removal with aluminum polychlorosulfate, whereas polyhydroxyaromatics are preferentially eliminated by ferric chloride [START_REF] Vilgé-Ritter | Removal of natural organic matter by coagulation-flocculation : a pyrolysis-GC-MS study[END_REF]. Another difficulty in identifying NOM destabilization mechanisms concerns the speciation of hydrolyzable metal salts used as coagulants. Even though, the aqueous chemistry of aluminum and iron based coagulants is now much better described, the formation of hydrolyzed metal species can be altered in the presence of -4 -NOM. Thus, the hydrolysis of ferric chloride is hindered to the trimer stage at both pH 5 and 7 [START_REF] Vilgé-Ritter | Removal of natural organic matter by coagulation-flocculation : a pyrolysis-GC-MS study[END_REF], while hydrolyzed aluminum species have been shown to be depolymerized by natural organic ligands [START_REF] Masion | Coagulation-flocculation of natural organic matter with Al salts : speciation and structure of the aggregates[END_REF].

The aim of this work was to investigate the destabilization mechanism of a model of a humic substance (MHS) with Al 13 polycations as a function of both pH and aluminum concentration. Al 13 polycations are formed during hydrolysis of most commercial polyaluminum chloride coagulants [START_REF] Wang | The nature of polynuclear OH-Al complexes in laboratoryhydrolyzed and commercial hydroxyaluminum solutions[END_REF][START_REF] Lartiges | Flocculation of colloidal silica with hydrolyzed aluminum : An 27 Al solid state NMR investigation[END_REF], whereas humic substances generally represent more than 50% of the dissolved NOM in lake and river waters [START_REF] Zumstein | Circulation of pedogenic and aquagenic organic matter in an eutrophic lake[END_REF]. MHS was obtained from auto-oxidation of catechol and glycine [START_REF] Jung | Synthesis of amino-phenolic humic-like substances and comparison with natural aquatic humic acids: A multi-analytical techniques approach[END_REF]. As the structure and basic chemical properties of natural humic substances are still a matter of debate, the definition and representativity of a synthetic model may always be discussed.

Nevertheless, extensive characterization of MHS and comparison with humic extracts from Moselle river water, revealed that such a synthesis yields organic molecules with elemental composition, titration features, spectral and fluorescence characteristics, similar to that of naturally occuring humic substances [START_REF] Jung | Synthesis of amino-phenolic humic-like substances and comparison with natural aquatic humic acids: A multi-analytical techniques approach[END_REF]. Furthermore, an Electron Energy Loss Spectroscopy (EELS) investigation of the coagulation of MHS and natural counterparts with a ferric salt, showed the same coagulation behavior for both organic colloids (Jung et al., in press), thus suggesting that MHS may represent a standard humic material suitable to compare the destabilization mechanism associated with various coagulants.

In this study, Magic-angle spinning (MAS) 27 Al NMR was used to investigate the fate of aluminum coagulant species during MHS aggregation, while crosspolarization magic-angle spinning (CP-MAS) 13 C NMR allowed to examine the nature of association reached between the aluminum polycations and the functional groups of MHS. Electrophoretic mobility provided valuable information about the overall charge of the MHS-coagulant association. Possible reconformation of the humic material may occur upon interaction with coagulant species. Fluorescence spectroscopy with pyrene as a polarity-sensitive probe was then used to investigate the conformational changes of MHS in presence of Al 13 polycations.

MATERIALS AND METHODS

Preparation of a model of a humic substance. MHS was synthesized by autooxidation from an aqueous equimolar mixture of catechol and glycine (0.03 mol/L in a 2 L flask) [START_REF] Jung | Synthesis of amino-phenolic humic-like substances and comparison with natural aquatic humic acids: A multi-analytical techniques approach[END_REF]. The reaction was conducted in the dark under constant stirring for 5 days. The temperature was maintained at 20°C and the pH was fixed at 7.9 with a phosphate buffer. The oxidative polymerization resulted in a dark-brown product which was purified by dialysis (6-8 kg/mol Spectra Por 6) against distilled water to yield a humic-like substance in a sodium form. MHS was then freeze-dried and stocked as a dry powder.

Humic substances are traditionally categorized into humins, humic acids, and fulvic acids, on the basis of their water solubility. At pH < 2, about 85% of our synthetic product is insoluble which defines MHS as a humic acid. Elemental analysis of MHS (Carlo Erba 1108 autoanalyser) indicated contents of 45.6% in C, 3.6% in H, 2.5% in N, and 48.3% in O. Such values are consistent with the mean composition of freshwater humic acids indicated in the literature [START_REF] Krasner | Three approaches for characterizing NOM[END_REF]. The total titrable charge of MHS (3.7 meq/g between pH 3.5-10.5) and the contents in carboxylic and weakly acidic groups (respectively 3.1 and 0.6 meq/g), are also in good agreement with the values reported for river water humic substances [START_REF] Christensen | Proton binding by gruwndwater fulvic acids of different age, origins, and structure modeled with the model V and nica-donnan model[END_REF]. However, even though humic molecules are known to aggregate, the use of a 6-8 kg/mol dialysis cut-off is expected to yield a material with a larger molecular weight than the 1-3 kg/mol generally reported for aquagenic humic material [START_REF] Beckett | Determination of molecular weight distributions of fulvic and humic acids using flow field-flow fractionation[END_REF]. Such a synthesis is also expected to yield organic polymeric colloids [START_REF] Swift | Macromolecular properties of humic substances: fact, fiction, and opinion[END_REF], rather than supramolecular assemblies [START_REF] Piccolo | The supramolecular structure of humic substances[END_REF]. Nevertheless, fluorescence monitoring of MHS synthesis suggested that the humic-like fluorescent compound was formed from the association of oligomeric quinonic species [START_REF] Jung | Synthesis of amino-phenolic humic-like substances and comparison with natural aquatic humic acids: A multi-analytical techniques approach[END_REF]. [START_REF] Bottero | Studies of hydrolyzed aluminum chloride solutions. 1. Nature of aluminum species and composition of aqueous solutions[END_REF]. The Al 13 polycation possesses a Keggin structure, which consists of a central tetrahedrally coordinated aluminum (Al IV ) surrounded by 12 edge-linked octahedral aluminum sites (Al VI ).

Preparation of

Preparation of suspensions and aggregation procedure. Suspensions were obtained by dissolving 30 mg of MHS in 1 L of deionised water, thus yielding a Total Organic Carbon (TOC) concentration of 12 mg/L. Such concentration is consistent with average TOC values usually reported for river waters [START_REF] Meybeck | Carbon, Nitrogen, and phosphorus transport by world rivers[END_REF]. NaHCO 3 (4 10 -3 mol/L) was also added to the synthetic water to provide pH and ionic strength buffers similar to that of Seine river water. Prior to coagulant injection, the pH of suspensions was adjusted to pH 6 or 8 with dropwise addition of 0.5 M HCl.

Aggregation of MHS with Al 13 polycations was performed in a standard 1 L glass beaker (90 mm diameter, 150 mm high) fitted with four Plexiglas baffles. Stirring was carried out with a 15 mm x 54 mm blade positioned at one-third the height of the reactor from the bottom. A stirring rate of 100 rpm, which corresponds to a spatially averaged velocity gradient G of 135 s -1 , was used for all the experiments. The required amount of Al 13 solution was added under agitation with a syringe at a point just below the free surface of the suspension. The time of injection was about 3 s. pH was not readjusted after coagulant addition as the drop in suspension pH resulting from further hydrolysis of the coagulant never exceeded 0.4 pH unit for the largest aluminum concentration used at pH 8. Stirring was continued at 100 rpm for 20 minutes, and at the end of mixing, the aggregated suspension was allowed to settle for 24 h in graduated Imhoff cones. The supernatant was then analyzed for its turbidity (Hach turbidimeter), TOC (Dorhman 190), and residual aluminum concentration (ICP-AES Jobin-Yvon 70 type B model with external calibration). The sediment was collected and dialysed against deionized water for 5 days to remove added ions. It was then freeze-dried for NMR studies. Preliminary 27 Al MAS NMR experiments indicated that the MHS-Al aggregates do not evolve significantly during settling and dialysis.

Electrophoretic mobility. The electrophoretic mobility of unsettled aggregates was determined with a Zetaphoremeter III (Sephy, France) equipped with a CCD camera.

Prior to the measurements, the supernatant of the settled suspensions was centrifuged at 2500 rpm for 10 minutes in a lab-centrifuge (Eppendorf 5804) to increase the concentration in particles. The centrifugate was then re-suspended in 50 ml of original supernatant before being pumped into the electrophoretic cell. This procedure may induce a slight compaction of the floc structure. However, such a rearrangement occurs at a much larger scale than that associated with the diffuse double layer. The velocity of particles located at the stationary layer was directly computed from a video analysis obtained at fixed time intervals under an 80 mV applied voltage.

Magic-angle spinning nuclear magnetic resonance (MAS-NMR). The MAS-NMR

spectra were recorded on a Bruker ASX 500 instrument in 4 mm zirconia rotors with polyimide endcaps. One-pulse 27 Al (I=5/2) spectra were recorded at 130.32 MHz, spinning at 14 kHz with recycle delays of 1 s and 400 transients. Small pulse widths (< π/2(2I+1) were used with duration of 0.5 s to obtain quantitative responses. { 1 H-13 C} cross-polarization CP/MAS-NMR spectra were obtained at 125.77 MHz, spinning at 10 kHz, with < π/2 1 H pulse of 8 s, contact times of 1 ms, recycle delays of 5 s and around 14000 transients. Line broadening procedure (100 Hz) was applied before Fourier transform, and the chemical shifts were quoted relative to Al(H 2 O) 6 3+ and TMS for 27 Al and 13 C respectively. Resonance intensities were obtained by integration for 27 Al and by decomposition into lorentzian components for 13 C.

Fluorescence measurements. One liter solutions containing 30 mg MHS and 336 mg NaHCO 3 in deionised water were first prepared and adjusted to either pH 6 or 8 with dropwise addition of 0.5 M HCL. 5 mL aliquots of MHS solution were then placed in glassware beakers and mixed with 5 µL of pyrene stock solution (10 -3 M pyrene (Aldrich 99+%) in ethanol (Carlo Erba 99.5%), thus yielding a pyrene concentration of the order of 1 µM for all fluorescence measurements. The volumetric content of ethanol introduced in the samples did not exceed 0.1% and does not influence the solubility of pyrene in samples. Al 13 coagulant was then injected, and the aggregated MHS-pyrene suspension was allowed to equilibrate for one hour. Prior to fluorescence measurements, the samples were re-homogeneized by over-hand shaking.

Steady-state fluorescence measurements were performed on a Fluorolog-2 spectrofluorometer (SPEX, Edison), using 0.5 mm slits for the excitation and emission monochromators. All spectra were recorded at room temperature. In order to compare the fluorescence intensities of all fluorescence spectra and to avoid equipment baseline time drift, the fluorescence was normalised with a 0.16 mg/L fluorescein solution buffered at pH 10, using an excitation wavelength of 488 nm with emission recorded from 450 to 550 nm. Pyrene excitation wavelength was set at 332 nm and the emission was collected between 350 and 450 nm. Even though MHS absorbance remained significant at the excitation wavelength, the background emission of the synthetic material was negligible and represented less than 2% of the total measured pyrene probe fluorescence. The polarity sensitive ratio I 1 /I 3 was calculated from the emission intensities at 372 (I 1 ) and 383 nm (I 3 ).

Preliminary blank experiments indicated negligible variations in MHS

background emission in presence of various Al 13 concentrations. It was also checked that pyrene fluorescence spectra were not affected by the addition of increasing concentrations of coagulants, and that the amount of Clanion contained in the aluminum coagulant solution had no quenching effect in the concentration range used. Furthermore, the emission spectra failed to reveal any excimer peak.

RESULTS AND DISCUSSION

Aggregation results. The effect of varying concentrations of Al 13 coagulant solution on MHS aggregation at both pH 6 and 8, is illustrated in figure 1. Three curves are shown, settled water turbidity, sediment volume, and residual aluminum concentration, each as a function of aluminum dosage. Figure 1a reveals that, at low aluminum concentration and for both pHs, the supernatant turbidity increases steadily with coagulant dosage to reach a maximum, and then decreases steeply upon further increase in aluminum concentration. The point where the extrapolated steep portion of the turbidity curve intersects the x-axis was chosen as the optimum coagulant concentration (OCC), and is indicated with an arrow on the graph. For our synthetic water, OCC values of about 2.4 10 -4 and 4.2 10 -4 mol/L were found at pH 6 and 8, respectively.

Excellent organic removal is then provided as more than 95% TOC is eliminated at both optimum coagulant dosages. At higher aluminum concentrations, the residual turbidity remains very low at pH 8, whereas it increases strongly above a 4.7 10 -4 mol/L coagulant concentration at pH 6 to reach a constant turbidity of about 12 NTU.

The drop in supernatant turbidity observed just before the OCC at pHs 6 and 8 corresponds to the formation of settleable aggregates as evidenced by a rapid build-up in sediment volume in this concentration range (figure 1b). Above the OCC, the sediment volume increases slightly at pH 8 to reach a plateau value, while it diminishes sharply at pH 6 as MHS is restabilized. The evolution of residual aluminum concentration follows the same general pattern as the supernatant turbidity curve (figure 1c): at low coagulant dosages, the residual aluminum concentration increases proportionally to the coagulant concentration, and then drops at the OCC to a similar 3.10 -5 mol/L value for both pHs. Such a behavior clearly reveals a strong association between MHS and hydrolyzed aluminum species. At pH 6, upon restabilization of the suspension, the residual aluminum concentration resumes its linear increase with coagulant dosage.

The difference in OCC concentration observed between pH 6 and 8 is the result of both an increased deprotonation of MHS functional groups and a decreased charge of coagulant species at pH 8. In the aluminum concentration range of OCCs, potentiometric titrations showed that the charge of the Al 13 polycation is about 6+ at pH 6, and 1+ at pH 8 [START_REF] Furrer | On the chemistry of the keggin Al13 polymer. I. Acid-base properties[END_REF]. It can then be calculated that at pH 6, the amount of charge brought by the coagulant species at OCC (~ 3.3 meq/g) is equivalent to the carboxylic acidity of the humic-like polymer (3.1 meq/g). On the other hand, at pH 8, the optimal coagulant demand represents a charge of 1.1 meq/g, i.e. about 3.3 times less than required to neutralize all the anionic groups available. This, at first, seems to substantiate previous literature indicating that different mechanisms are responsible for humic substances removal according to pH (e.g. [START_REF] Dempsey | The coagulation of humic substances by means of aluminum salts[END_REF]. The 63 ppm signal can be unambiguously assigned to the central tetrahedrally coordinated aluminum in the Al 13 polycation [START_REF] Bottero | Studies of hydrolyzed aluminum chloride solutions. 1. Nature of aluminum species and composition of aqueous solutions[END_REF]. The strong slightly asymetric 3 ppm signal falls within the six coordinate chemical shift region of aluminum and is similar to values reported for the dimer Al 2 (OH) 2 4+ [START_REF] Masion | Coagulation-flocculation of natural organic matter with Al salts : speciation and structure of the aggregates[END_REF], and for monomeric and oligomeric species of aluminum complexed with low molecular weight organic acids [START_REF] Masion | Chemistry and structure of Al(OH)/Organic precipitates. A small angle X-ray scattering study. 3. Depolymerization of Al 13 polycation by organic ligands[END_REF]. However, closer examination of the spectra reveals that the maximum of this resonance shifts gradually from 2 to 7 ppm with increasing aluminum concentration and pH (figure 3a). Previous NMR investigations of fresh Al 13 precipitates have attributed such a shift to octahedral aluminum in polymeric species of Al 13 units of increasing degree of polymerization [START_REF] Bradley | The structure of Al gels formed through the base hydrolysis of Al 3+ aqueous solutions[END_REF][START_REF] Lartiges | Flocculation of colloidal silica with hydrolyzed aluminum : An 27 Al solid state NMR investigation[END_REF]. Recent EXAFS and NMR studies of Ga 13 -11 -aggregates have shown that the association of Keggin polycations occurs through double corner sharing coordinated water molecules (Lartiges et al., in preparation). In comparison with isolated Al 13 polycations, an increase in symmetry and deshielding is then expected for the aluminum octahedra upon the formation of Al 13 oligomers. The decrease of Full Width at Half Height (FWHH) of the octahedral resonance with aluminum concentration is consistent with such a view (figure 3b).

Fate of coagulant species.

The peaks at 64.5 and 33 ppm have been previously identified in solutions of Al 13 tridecamers aged at high temperature [START_REF] Fu | Aging processes of alumina sol-gels : characterization of new aluminum polyoxycations by 27 Al NMR spectroscopy[END_REF][START_REF] Allouche | Al 30 : A giant aluminum polycation[END_REF], and in powders obtained from AlCl 3 /urea hydrolyzed sols [START_REF] Wood | Hydrolysis of aluminium. Are all gels created equal ? Better Ceram[END_REF]. The 33 ppm signal, indicative of a five-coordinate Al [START_REF] Wood | Hydrolysis of aluminium. Are all gels created equal ? Better Ceram[END_REF], is not stable in solution and is necessarily generated during the freeze-drying of sediments. Assignment of the 64.5 ppm resonance is controversial as it has been attributed (i) to the tetrahedral site of a 12 aluminum polycation (Al VI /Al IV = 11) arising from the loss of one octahedral Al from an Al 13 unit [START_REF] Fu | Aging processes of alumina sol-gels : characterization of new aluminum polyoxycations by 27 Al NMR spectroscopy[END_REF], (ii) to an Al 13 Keggin structure capped with 1 to 3 AlO 6 octahedra [START_REF] Allouche | Al 30 : A giant aluminum polycation[END_REF]. Nevertheless, both polycations have been described as intermediate species formed during the oligomerization of Al 13 units, which is in accordance with the increase of the 64.5 ppm peak area with Al concentration (figure 2).

A rough estimate of the Al IV to Alt contributions reveals that the Al IV /Alt ratio is greater than 8 in the aluminum concentration range investigated by solid state 27 Al NMR (figure 3c). Therefore, Al 13 polymers and aluminum polycations with a Al VI /Al IV < 12, are the dominating species within dialyzed coagulated MHS. Previous reports indicated that low molecular weight organic acids hinder aluminum hydrolysis [START_REF] Masion | Coagulation-flocculation of natural organic matter with Al salts : speciation and structure of the aggregates[END_REF] and depolymerize preformed Al 13 polycations [START_REF] Molis | Chemical and structural transformation of aggregated Al13 polycations, promoted by salicylate ligand[END_REF][START_REF] Amirbahman | Kinetics and mechanism of ligand promoted decomposition of the Keggin Al 13 polymer[END_REF]. Our results suggest that, in the OCC range and in the timescale of the experiment, the Al 13 coagulant species seem to be stable in presence of MHS, which is consistent with previous evidence of Al 13 polycations found in organic soil horizons [START_REF] Hunter | Evidence of phytotoxic hydroxy-aluminum polymer in organic soil horizons[END_REF]. Nevertheless, some depolymerization of Al 13 polymers may occur as the Al IV /Alt ratio is observed to decrease at lower coagulant dosages. Attempts to determine the speciation of aluminum with liquid state 27 Al NMR at low aluminum concentration were unsuccessful, thus indicating that the aluminum species are strongly associated with the humic material.

Interestingly, Figure 3 reveals some kind of continuity as a function of aluminum concentration between values obtained at pH 6 and 8 for percent of tetrahedral aluminum, FWHH of octahedrally coordinated species, and octahedral aluminum chemical shift. This suggests that the size and degree of polymerization of Al 13 units increase regularly with coagulant concentration irrespective of pH. In other terms, 27 Al NMR does not reveal any contrasting behavior in the formation of coagulant species between pH 6 and 8, or low and high aluminum concentrations. Thus, the formation of a voluminous mass of aluminum hydroxyde precipitate usually reported in the sweep-flocculation domain (e.g. [START_REF] Edwards | Removing color caused by humic acids[END_REF] is not detected.

Interaction between coagulant species and MHS functional groups. The CP-MAS

13 C NMR spectra of MHS coagulated with various aluminum concentrations at pH 6 and 8 are shown in figure 4. All spectra display major signals at about 176, 145, and 117 ppm which are easily assigned to carboxylic carbon, phenolic ring carbon (aromatic-oxygen bound), and aromatic ring carbon, respectively [START_REF] Inbar | Solid state carbon 13 nuclear magnetic resonance and infrared spectroscopy of composed organic matter[END_REF]. In addition, the original non coagulated MHS exhibits a small peak at about 85 ppm, which is characteristic of aliphatic carbon singly bonded to oxygen or nitrogen [START_REF] Stuermer | Investigation of seawater and terrestrial humic substances with carbon -13 and proton nuclear magnetic resonance[END_REF].

The main characteristic of the CP-MAS technique is that the magnetization is transferred to 13 C from neighboring protons 1 H through dipolar coupling which is strongly distance dependent: in other terms, 13 C that are close to protons generally cross-polarize more efficiently than 13 C that are some distance away from a proton.

Applied to MHS-Al aggregates, 13 C CP-MAS NMR may be used to probe the presence of highly hydrated coagulant species in the vicinity of a given functional group. Thus, at pH 6, the relative abundance of the peak at 176 ppm increases with aluminum concentration to become predominant for [Al] ≥ 2.6 10 -4 mol/L. Such a result is then in agreement with the picture of positive coagulant species anchored on negatively charged carboxylic groups. Likewise, the 117 ppm resonance, poorly resolved at low coagulant concentration, is enhanced at higher aluminum dosage. Previous studies have shown that MHS carboxylic groups originate in part from the cleavage of catechol aromatic rings during oxydative polymerization [START_REF] Furrer | On the chemistry of the keggin Al13 polymer. I. Acid-base properties[END_REF]. Therefore, it is not surprising that the evolutions of carboxyl and aromatic carbon resonances can be related. Interestingly, the carboxylic to aromatic C ratio increases linearly with aluminum concentration up to the COC as illustrated in figure 5a. Finally, just before restabilization of aggregates, the 117 ppm signal increases strongly. This suggests that phenolic groups are available for association with Al 13 polycations in presence of excess coagulant.

At pH 8, the 13 C CP-MAS NMR spectra obtained above the OCC closely resemble the spectra at pH 6 with similar proportions of carboxyl, phenolic ring, and aromatic ring carbons in the optimal concentration range. However, at pH 8, the intensities of carboxyl, phenolic, and aromatic peaks are almost equivalent at low coagulant dosages (figure 4), with the aromatic peak significantly dominating at [Al] = 4.10 -4 mol/L. This suggests that the ionized functional groups of MHS do not interact with Al 13 polycations in the same order according to pH. Thus, the enhanced aromatic carbon signal at pH 8, indicates that the aluminum hydrolyzed species preferentially associate with phenolic moieties in the low coagulant concentration range, and then with carboxylic functional groups at higher aluminum dosages. The carboxylic to aromatic carbon ratios obtained at pH 8 further illustrate such a view (figure 5b).

Electrophoretic mobility of Al-MHS aggregates.

The electrophoretic mobilities of MHS coagulated at pH 6 and 8 with various concentrations of Al 13 polycations, are shown in figure 6. Up to the OCC, the aggregates present a constant negative mobility at both pH 6 and 8 although the humic-like substance coagulated well in this concentration range. Therefore, in contrast to oxide or clay colloidal particles aggregated with metal hydrolyzable coagulants (e.g. [START_REF] James | Charge reversal coagulation of colloidal dispersions by hydrolysable metal ions[END_REF], the mobility curve of MHS aggregates provides no indication of the optimal coagulation zone. This implies that the destabilisation mechanism operating at pH 6 can not correspond to a simple charge neutralization of MHS with Al 13 polycations. Above OCC, the electrophoretic mobilities reduce to less negative values until charge reversal of aggregates. Comparison with the Jar test results indicates that at pH 6, the restabilization of aggregates begins with nearly neutral aggregates, whereas at pH 8, the lower positive electrophoretic mobilities obtained in presence of excess coagulant seem insufficient to trigger MHS redispersion. This effect can be ascribed to a difference in binding strength between the coagulant species formed at pH 6 and 8. Indeed, Al 13 polycations, charged 6+ at pH 6 should be more effective in changing the conformation of humic chains, than the lesser charged coagulant species formed at pH 8. Such a reorganization of MHS chains around positive coagulant species would then yield the negative electrophoretic mobilities observed in the optimal removal range at both pH 6 and 8.

Pyrene fluorescence in MHS coagulated suspensions. Figure 7 illustrates the aluminum concentration dependence of the I 1 /I 3 fluorescence intensity ratio at pHs 6 and 8. In order to facilitate the discussion, electrophoretic mobility results are also plotted on the same graph. The intensity ratio of the first and third vibronic peaks in the pyrene fluorescence spectrum is known to provide a good solvent polarity sensor at the local scale [START_REF] Jung | Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems[END_REF]. In pure water, I 1 /I 3 is about 1.9, while it is much lower in an apolar medium (e.g. I 1 /I 3 ~ 0.6 in hexane). The recorded value of I 1 /I 3 is then an average of that reported for pyrene associated with MHS and pyrene in the bulk aqueous phase.

In the original MHS suspension, I 1 /I 3 is equal to 1.8 which indicates an average solvent environment of pyrene slightly less polar than water. Upon addition of the aluminum coagulant, I 1 /I 3 decreases at both pH 6 and 8 to reach a minimum for [Al] = 1.4 10 -4 mol/l. This suggests that the pyrene probe has been solubilized within a more hydrophobic environment. Such a behavior can be explained by (i) an increased partition of pyrene into newly created hydrophobic domains, and/or (ii) a reorganization of humic chains in the vicinity of previously sorbed pyrene molecules. However, as the fluoroprobe is expected to be largely associated with MHS prior to the addition of Al 13 polycations [START_REF] Chin | Binding of pyrene to aquatic and commercial humic substances: the role of molecular weight and aromaticity[END_REF][START_REF] Kopinke | Sorption of pyrene to dissolved humic substances and related model polymers. 1. Structure-property correlation[END_REF], the second hypothesis is favored.

The minimum values measured for I 1 /I 3 , about 1.75 at both pH 6 and 8, are in close agreement with I 1 /I 3 intensity ratios found for Latahco silt loam humic acid in the presence of MgCl 2 [START_REF] Engebretson | The effect of molecular size on humic acid associations[END_REF].

Further increase in coagulant dosage reverts the evolution of I 1 /I 3 , and the initial environmental polarity around pyrene molecules is restored for aluminum concentrations of 4.4 10 -4 and 7 10 -4 mol/l at pHs 6 and 8, respectively. This peculiar phenomenon can be compared with a demicellization process at the macromolecule scale. As increasing amounts of Al 13 polycations are added to the MHS suspension, the coagulant species are expected to compete for the negatively charged moieties of humic chains. Therefore, instead of being attached onto functional groups of more than one chain thus determining intramolecular bridging links, the positive coagulant species become associated with individual carboxylic and phenolic groups, which eventually results in electrostatic repulsion between humic chains. The gradual reduction in electrophoretic mobility of MHS-Al aggregates to zero in this range of concentration, corroborates such a view.

CONCLUSION

In the pH range relevant to drinking water treatment, the removal of humic substances is generally described by at least two destabilization mechanisms according to pH and coagulant concentration. Contrary to this widely accepted view, our results suggest a similar MHS/Al 13 aggregation at both pHs 6 and 8. A comparable conclusion was reached by Vilgé-Ritter et al. (1999b) in her combined EXAFS and SAXS study of NOM destabilization with ferric chloride at pH 5.5 and 7.5. -24 -Figure 6 -25 -

FIGURE CAPTIONS

Figure 7b

  Figure 2 shows the 27 Al MAS NMR spectra obtained for MHS coagulated at both pH 6 and 8 with different aluminum concentrations. All spectra exhibit two prominent resonances at about 3 and 63 ppm, and two weaker less resolved peaks at about 33 and 64.5 ppm.

Figure 1 .

 1 Figure 1. Evolution as a function of aluminum concentration of (a) residual turbidity (b) Sediment volume (c) Residual aluminum concentration in supernatant. pH 6 filled symbols -pH 8 open symbols.

Figure 2 .

 2 Figure 2. 27 Al MAS NMR spectra of freeze-dried MHS coagulated with different concentrations of aluminum at pHs 6 and 8.

Figure 3 .

 3 Figure 3. Evolution as a function of aluminum concentration of (a) Peak chemical shift of 27 Al octahedral resonances. (b) Full Width at Half Height for octahedrally coordinated species. (c) Percentage of Al IV /Alt ratio. pH 6 (filled circles) and at pH 8 (open circles).

Figure 4 .

 4 Figure 4. 13 C CP MAS NMR spectra of freeze-dried MHS coagulated with various aluminum concentrations at pHs 6 and 8.

Figure 5 .

 5 Figure 5. Intensity ratio of carboxylic carbon and aromatic carbon peaks calculated from 13 C NMR spectra of coagulated MHS (a) pH 6 and (b) pH 8.

Figure 6 .

 6 Figure 6. Evolution of the electrophoretic mobility of MHS-Al aggregates as a function of Al concentration at pHs 6 and 8.

Figure 7 .

 7 Figure 7. Evolution of pyrene I 1 /I 3 ratio intensity and electrophoretic mobility of MHS-Al aggregates. (a) pH 6, (b) pH 8.

Figure 1

 1 Figure 1Fig. 1. (a) Turbidity removal of 30mg/L of humic acid. (b) Sediment volume of aggregates after 24 hours settling. (c) Residual aluminum in supernatant. All parameters are measured as a function of aluminum dose and pH. Filled circles, diamonds and squares show variations at pH 6. Open circles, diamonds and squares show variations at pH 8.

  Figure 5

Al 13 stock solution. The

	Al 13 coagulant solution was prepared by
	dissolving analytical grade AlCl 3 .6H 2 O (Aldrich) in deionized water (MilliQ 18.5 M),
	and then by adding dropwise NaOH 0.45 M solution (0.4 mL/min) under vigorous
	stirring to an hydrolysis molar ratio OH/Al of 2.25 and a total Al concentration of 0.1
	M. Previous 27 Al NMR investigations revealed that such synthesis conditions
	essentially yield AlO 4 Al 12 (OH) 24 (H 2 O) 12 isolated polycations in the partially
	hydrolyzed solution

ACKNOWLEDGMENTS

This work was supported from funds provided by Suez-Lyonnaise des Eaux, France. This support is gratefully acknowledged. V.K. and B.S.L. thank CRPG-CNRS, Nancy France for ICP-AES analysis, Bernadette Gérard for help with the synthesis of MHS at CPB-CNRS, Nancy, France and Ka Yan Leung for help with the electrophoretic measurements.

Until a consensus can be reached regarding the structure and conformation of humic substances, any investigation carried out with a synthetic anologous of humic material will remain arguable. However, our study suggests that a reorganization of anionic humic chains occurs upon association with the positively charged coagulant species. Such a conclusion is consistent with humic acids viewed as soft and permeable structures [START_REF] Duval | Humic substances are soft and permeable: evidence from their electrophoretic mobilities[END_REF]. It is also in accordance with previous EELS results, obtained with both MHS and natural humic substances, suggesting that the optimal coagulant concentration depends on the interplay between the rate of collisions of humic colloids/coagulant species, and the rate of organic material reconformation [START_REF] Jung | Synthesis of amino-phenolic humic-like substances and comparison with natural aquatic humic acids: A multi-analytical techniques approach[END_REF]. In that case, for a given collision rate, larger molecular weight material with lower reconformation rate, should be removed prior to lower molecular weight organic colloids.