Clifford W Fong
email: cwfong@internode.on.net

Role of free radicals in PARP inhibition of cervical carcinogenesis: PARylation of HeLa cells, and inhibition of hPARP-1 enzyme

Keywords: PARP, PARP inhibitors, PARylation, cervical cancer HeLa cells, oxidative stress

Role of free radicals in PARP inhibition of cervical carcinogenesis: PARylation of HeLa cells, and inhibition of hPARP-1 enzyme

Introduction

PARP (Poly (ADP-ribose) polymerase) is a protein family involved in a number of cellular processes such as DNA repair, genomic stability, and programmed cell death. PARP's main function is to detect and initiate an immediate cellular repair response to metabolic, chemical, oxidative stress, or radiation-induced single-strand DNA breaks. PARylation (protein poly ADPribosylation) is a widespread post-translational modification at DNA lesions catalyzed by PARPs. This modification regulates a number of biological processes particularly DNA damage response but also transcriptional regulation, apoptosis, and mitosis. PARP can function as a DNA damage sensor activated by DNA lesions, thereby forming PAR chains that serve as a docking platform for DNA repair factors. [1,[START_REF] Hegedus | Inputs and outputs of poly(ADP-ribosyl)ation: Relevance to oxidative stress[END_REF] PARP is a key facilitator of DNA damage repair and has a key role in tumorigenesis associated with dysfunctional DNA repair pathways. Transcript, protein, and enzyme activity of PARP is increased in breast cancer, ovarian cancer, hepatocellular cancer, colorectal cancer, and leukemia. Treating cancers by targeting PARP through increasing tumour sensitivity to chemotherapeutic agents and also by inducing "synthetic lethality" is now common in the clinic. PARP inhibitors have been shown to be successful in the treatment of various cancers, attributed to impaired DNA repair of cancerous cells. It has been shown that PARP inhibition specifically kills BRCA1 and BRCA2 mutant cells, demonstrating synthetic lethality of PARP inhibition with the homologous recombination defect. It is thought that inhibitor-inactivated PARP-1 is trapped on DNA, and that trapped {PARP-1:DNA} complexes are cytotoxic. In addition, it is thought that loss of PARylation impairs early recruitment of both BRCA1 and BRCA2 to DNA lesions, suggesting that the PARylation directly contributes to homologous recombination. [START_REF] Wh Hou | Poly-ADP ribosylation in DNA damage response and cancer therapy[END_REF][START_REF] Liu | The role of poly ADP-ribosylation in the first wave of DNA damage response[END_REF][START_REF] Lupo | Inhibition of poly(ADP-ribosyl)ation in cancer: Old and new paradigms revisited[END_REF][START_REF] Mangerich | How to kill tumor cells with inhibitors of poly(ADP-ribosyl)ation[END_REF][START_REF] Sisay | PARP inhibitors as potential therapeutic agents for various cancers: focus on niraparib and its first global approval for maintenance therapy of gynaecologic cancers[END_REF][START_REF] Murai | Trapping of PARP1 and PARP2 by clinical PARP inhibitors[END_REF][START_REF] Kn Dziadkowiec | PARP inhibitors: review of mechanisms of action and BRCA1/2 mutation targeting[END_REF] PARP inhibitors are clinically effective in a number of tumour types, including platinumsensitive epithelial ovarian cancer, breast cancer with mutation in BRCA-1 or BRCA-2, and prostate cancer. Olaparib is a PARP inhibitor which has been approved as a monotherapy for the treatment of patients with germline BRCA mutated advanced ovarian cancer who have been treated with three or more prior lines of chemotherapy. Olaparib has been approved in Europe for maintenance therapy in both germline and somatic BRCA-mutant platinum-sensitive ovarian cancer. Rucaparib has been approved for previously treated BRCA-mutant ovarian cancer. Niraparib, a PARP-1 and PARP-2 inhibitor, is approved for epithelial ovarian, fallopian tube, and primary peritoneal cancer.

PARPs are also involved in oxidative stress, cell death, and oxidative-stress-related diseases. Oxidative stress can cause DNA breaks which results in activation of the DNA nick sensor enzyme PARP-1. Normal PARP activation facilitates DNA repair, but extensive PARP activation induces mitochondrial transition and mitochondrial damage that culminates in cell death. Mitochondrial dysfunction is strongly associated with PARP-mediated apoptotic cell death. PARP inhibition converts necrosis to apoptosis. The majority of PARP activity is located in the nucleus, but some PARP activity is found in the cytoplasm. [START_REF] Bai | Biology of Poly(ADP-Ribose) Polymerases: The Factotums of Cell Maintenance[END_REF] Inhibition or depletion of PARP leads to not only an increase in DNA damage, but also an elevation in the levels of reactive oxygen species (ROS). Oxidative stress is also increased by PARP inhibition and mediates the antitumor effect. The antioxidant N-acetylcysteine (NAC) attenuated the induction of DNA damage and the proliferation by PARP inhibition. NADPH oxidases 1 and 4 were upregulated by PARP inhibition and were partially responsible for the induction of oxidative stress. Thus in addition to compromising the repair of DNA damage, PARP inhibition also exerts an extra antitumor effect by elevating oxidative stress in ovarian cancer cells. [START_REF] Hou | Increased oxidative stress mediates the antitumor effect of PARP inhibition in ovarian cancer[END_REF][START_REF] Mathews | PARP-1 Inhibition Prevents Oxidative and Nitrosative Stress-Induced Endothelial Cell Death via Transactivation of the VEGF Receptor 2[END_REF] Hypoxic cells with decreased homologous recombination (HR) protein expression showed increased clonogenic killing following chemical inhibition of PARP1. Selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality" has been demonstrated. [START_REF] Chan | Contextual Synthetic Lethality of Cancer Cell Kill Based on the Tumor Microenvironment[END_REF][START_REF] Leszczynska | Targeting Tumour Hypoxia with PARP Inhibitors: Contextual Synthetic Lethality[END_REF] PARP inhibitors can potentiate the effects of radiotherapy and chemotherapy in cancer cells. Inhibition of PARP activity can sensitize hypoxic cancer cells and the combination of ionizing radiation with PARP inhibitors can improve the therapeutic ratio of radiotherapy. [START_REF] Sk Liu | A novel poly(ADPribose) polymerase inhibitor, ABT-888, radiosensitizes malignant human cell lines under hypoxia[END_REF] Olaparib is a potent radiosensitizer for head and neck cancer therapy. [START_REF] Tinhofer | The PARP inhibitor olaparib (AZD2281) as potent radiosensitizer of head and neck cancer cells[END_REF] Hypoxia and ease of reaction with ionizing radiation are hallmarks of free radical drug formation in chemoradiotherapy regimes. Drug-induced oxidative stress is implicated as a mechanism of toxicity in numerous tissues and organ systems. [START_REF] Fong | The extravascular penetration of tirapazamine into tumours: a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues and the potential use of cucurbiturils to facilitate delivery[END_REF][START_REF] Fong | Platinum based radiochemotherapies: Free radical mechanisms and radiotherapy sensitizer[END_REF][START_REF] Fong | Platinum anti-cancer drugs: Free radical mechanism of Pt-DNA adduct formation and anti-neoplastic effect[END_REF][START_REF] Fong | The role of free radicals in the effectiveness of anti-cancer chemotherapy in hypoxic ovarian cells and tumours[END_REF][START_REF] Fong | Role of stable free radicals in conjugated antioxidant and cytotoxicity treatment of triple negative breast cancer[END_REF][START_REF] Fong | Cucurbiturils as potential free radical chemoradiosensitizers for enhanced cisplatin treatment of cancers: a quantum mechanical study[END_REF][START_REF] Wardman | Electron transfer and oxidative stress as key factors in the design of drugs selectively active in hypoxia[END_REF] The metabolism of a drug may generate a reactive intermediate that can reduce molecular oxygen directly to generate ROS. [START_REF] Deavall | Drug-Induced Oxidative Stress and Toxicity[END_REF] Many currently prescribed antineoplastic drugs induce high levels of free radicals and OS, with patients showing ROS-induced lipid peroxidation in their plasma, have reduced blood levels of vitamin E, vitamin C and β-carotene, and decreased tissue glutathione levels. Many anticancer drugs are also capable of generating ROS such as superoxide, typically by redox cycling with oxygen in mitochondrial systems or in the endoplasmic reticulum. These drugs can be readily bioreduced by various enzymes, such as NADPH-cytochrome P450 reductases and FADH 2 etc. [START_REF] Ka Conklin | Chemotherapy-Associated Oxidative Stress: Impact on Chemotherapeutic Effectiveness[END_REF][START_REF] Sinha | Free radicals in anticancer drug pharmacology[END_REF][START_REF] Kappus | Overview of enzyme systems involved in bio-reduction of drugs and in redox cycling[END_REF][START_REF] Kovacic | Unifying mechanism for anticancer agents involving electron transfer and oxidative stress: clinical implications[END_REF][START_REF] Sainz | Radical Decisions in Cancer: Redox Control of Cell Growth and Death[END_REF][START_REF] Gorrini | Modulation of oxidative stress as an anticancer strategy[END_REF] Amongst the common antineoplastic drugs, the severity of oxidative stress varies from: (a) very high, anthracyclines: (b) moderately high, Pt-complexes, alkylating agents, epipodophyllotoxins (c) low, purines/pyrimidines, antimetabolites, taxanes, vinca alkaloids. [START_REF] Ka Conklin | Chemotherapy-Associated Oxidative Stress: Impact on Chemotherapeutic Effectiveness[END_REF][START_REF] Sinha | Free radicals in anticancer drug pharmacology[END_REF][START_REF] Kappus | Overview of enzyme systems involved in bio-reduction of drugs and in redox cycling[END_REF][START_REF] Kovacic | Unifying mechanism for anticancer agents involving electron transfer and oxidative stress: clinical implications[END_REF][START_REF] Sainz | Radical Decisions in Cancer: Redox Control of Cell Growth and Death[END_REF][START_REF] Gorrini | Modulation of oxidative stress as an anticancer strategy[END_REF][START_REF] Liu | Increased Oxidative Stress as a Selective Anticancer Therapy, Oxidative Medicine and Cellular Longevity[END_REF] PARP inhibitors also involve free radicals and oxidative stress in their chemotherapeutic regimes. [START_REF] Hou | Increased oxidative stress mediates the antitumor effect of PARP inhibition in ovarian cancer[END_REF][START_REF] Ga Czapski | Effect of poly(ADP-ribose) polymerase inhibitors on oxidative stress evoked hydroxyl radical level and macromolecules oxidation in cell free system of rat brain cortex[END_REF] Figure 1 schematically illustrates the various processes known to be involved in the inhibition of PARP-1 enzymes and PARylation and accompanying oxidative stress induced by PARP activation.

We have previously shown that an equation of the general form shown below, eq 1, applies to cell membrane transport processes for various drugs as well as drug-enzyme binding interactions. [START_REF] Fong | The extravascular penetration of tirapazamine into tumours: a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues and the potential use of cucurbiturils to facilitate delivery[END_REF][START_REF] Fong | Drug discovery model using molecular orbital computations: tyrosine kinase inhibitors[END_REF][START_REF] Fong | Permeability of the Blood-Brain Barrier: Molecular Mechanism of Transport of Drugs and Physiologically Important Compounds[END_REF][START_REF] Fong | The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance[END_REF][START_REF] Fong | A novel predictive model for the anti-bacterial, anti-malarial and hERG cardiac QT prolongation properties of fluoroquinolones[END_REF][START_REF] Fong | Statins in therapy: Cellular transport, side effects, drug-drug interactions and cytotoxicity -the unrecognized role of lactones[END_REF][START_REF] Fong | Physiology of ionophore transport of potassium and sodium ions across cell membranes: Valinomycin and 18-Crown-6 Ether[END_REF][START_REF] Fong | Statins in therapy: Understanding their hydrophilicity, lipophilicity, binding to 3-hydroxy-3-methylglutaryl-CoA reductase, ability to cross the blood brain barrier and metabolic stability based on electrostatic molecular orbital studies[END_REF]

Transport or Binding = ΔG desolv,CDS + ΔG lipo,CDS + Dipole Moment + Molecular Volume or Electron Affinity

This equation 1 uses the free energy of water desolvation (ΔG desolv,CDS ) and the lipophilicity free energy (ΔG lipo,CDS ) where CDS represents the non-electrostatic first solvation shell solvent properties, may be a better approximation of the cybotactic environment around the drug approaching or within the protein receptor pocket, or the cell membrane surface or the surface of a drug transporter, than the bulk water environment outside the receptor pocket or cell membrane surface. The CDS includes dispersion, cavitation, and covalent components of hydrogen bonding, hydrophobic effects. Desolvation of water from the drug (ΔG desolv,CDS ) before binding in the receptor pocket is required, and hydrophobic interactions between the drug and protein (ΔG lipo,CDS ) is a positive contribution to binding. ΔG lipo,CDS is calculated from the solvation energy in n-octane. In some biological processes, where biological redox processes may be occurring, and the influence of molecular volume is small, the electron affinity EA has been included in place of the molecular volume, MV. In other processes, the influence of some of the independent variables is small and can be eliminated to focus on the major determinants of biological activity.

We have recently used this model to develop a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues of tirapazmine and the effect on the extravascular penetration of tirapazamine into tumours. [START_REF] Fong | The extravascular penetration of tirapazamine into tumours: a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues and the potential use of cucurbiturils to facilitate delivery[END_REF] It was found that the multiparameter model of the diffusion, antiproliferative assays IC 50 and aerobic and hypoxic clonogenic assays for a wide range of neutral and radical anion forms of tirapazamine (TPZ) analogues showed: (a) extravascular diffusion is governed by the desolvation, lipophilicity, dipole moment and molecular volume, similar to passive and facilitated permeation through the blood brain barrier and other cellular membranes, (b) hypoxic assay properties of the TPZ analogues showed dependencies on the electron affinity, as well as lipophilicity and dipole moment and desolvation, similar to other biological processes involving permeation of cellular membranes, including nuclear membranes, (c) aerobic assay properties were dependent almost exclusively on the electron affinity, consistent with electron transfer involving free radicals being the dominant species.

Results

The general equation ( 1) has been applied to the IC 50 and EC 50 data from the Table for the hexahydrobenzonaphthyridinone PARP inhibitors. All IC 50 and EC 50 data are from the same study and laboratory source [START_REF] Torrisi | Discovery and SAR of novel, potent and selective hexahydrobenzonaphthyridinone inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1)[END_REF] (see Figure 2) to minimize experimental data error. NB: Raw MV values have been normalized by factor of 0.1, and raw EA values normalized by 10.0 to allow direct comparison of coefficients in the equations.

Since there is literature evidence [START_REF] Hou | Increased oxidative stress mediates the antitumor effect of PARP inhibition in ovarian cancer[END_REF][START_REF] Ka Conklin | Chemotherapy-Associated Oxidative Stress: Impact on Chemotherapeutic Effectiveness[END_REF][START_REF] Sinha | Free radicals in anticancer drug pharmacology[END_REF][START_REF] Kappus | Overview of enzyme systems involved in bio-reduction of drugs and in redox cycling[END_REF][START_REF] Kovacic | Unifying mechanism for anticancer agents involving electron transfer and oxidative stress: clinical implications[END_REF][START_REF] Sainz | Radical Decisions in Cancer: Redox Control of Cell Growth and Death[END_REF][START_REF] Gorrini | Modulation of oxidative stress as an anticancer strategy[END_REF][START_REF] Liu | Increased Oxidative Stress as a Selective Anticancer Therapy, Oxidative Medicine and Cellular Longevity[END_REF][START_REF] Ga Czapski | Effect of poly(ADP-ribose) polymerase inhibitors on oxidative stress evoked hydroxyl radical level and macromolecules oxidation in cell free system of rat brain cortex[END_REF] that the free radical form of various anticancer drugs involve the free radical form of the drug (as well as some PARP inhibitors) in their chemotherapy, the experimental data in the Table has been tested against the free radical form, as well as the neutral (or protonated) form of the drugs. Applying the general equation ( 1 However it is possible that PARylation in the HeLa cells may involve non-redox processes, and that inhibition occurs through the neutral or protonated at physiological pH levels. This is analysed in eq 3: Comparison of eq 3 with eq 4 shows that DM has greater influence for eq 3 for the in vitro hPARP-1 data, but otherwise the equations are very similar in terms of water desolvation, lipophilicity and molecular volumes of the inhibitors in the cellular environment compared to the in vitro environment.

EC

Discussion

Figure 1 illustrates the various processes involved in the inhibition of PARP-1 enzymes and PARylation of HeLa cells and the accompanying oxidative stress induced by PARP activation.

Oxidative stress is also increased by PARP inhibition and mediates the antitumor effect. The IC 50 data for the in vitro inhibition of the hPARP-1 enzyme can be taken as a close substitute of the inhibition of PARP-1 in cells (if membrane transport processes are comparatively insignificant), while the EC 50 data for the inhibition of the HeLa cells would include the inhibition of the PARP-1 enzyme preceding the inhibition of PARylation (and any membrane transport processes). It has been recently shown it is the kinetics of poly(ADP-ribosyl)ation, but not PARP-1 itself, that determines the cell fate in response to DNA damage in vitro and in vivo.

Thus the homeostasis and dynamics of PAR formation is critical to the cells and organisms fate in coping with acute DNA damage, whereas the full PARylation response is essential for organism survival under genotoxic stress. PARylation involves the PARP catalysed transfer of the ADP-ribose moiety from β-nicotinamide-adenine-dinucleotide (β-NAD + ) to acceptor amino acid residues of target proteins and the creation of long PAR chains. [START_REF] Schuhwerk | Kinetics of poly(ADPribosyl)ation, but not PARP1 itself, determines the cell fate in response to DNA damage in vitro and in vivo[END_REF] The EC 50 and IC 50 data of Torrisi [START_REF] Torrisi | Discovery and SAR of novel, potent and selective hexahydrobenzonaphthyridinone inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1)[END_REF] for the hexahydrobenzonaphthyridinone PARP inhibitors are valuable since it allows comparison of the cellular processes involved in PARylation of HeLa cells with the in vitro inhibitor binding to the hPARP-1 enzyme. The IC 50 inhibitory data could involve the neutral or protonated forms of the hexahydrobenzonaphthyridinones, since many of the inhibitors are likely to be partially protonated at physiological pH levels. There is strong evidence from this study, and literature sources [START_REF] Bai | Biology of Poly(ADP-Ribose) Polymerases: The Factotums of Cell Maintenance[END_REF][START_REF] Hou | Increased oxidative stress mediates the antitumor effect of PARP inhibition in ovarian cancer[END_REF][START_REF] Mathews | PARP-1 Inhibition Prevents Oxidative and Nitrosative Stress-Induced Endothelial Cell Death via Transactivation of the VEGF Receptor 2[END_REF], that oxidative stress may be involved in PARylation processes, and that the anion radical form of the inhibitors could be involved in the EC 50 PARylation processes in the HeLa cells. These possibilities have been tested in the results for equations 2 -4 using the neutral, protonated or anion radical form of the neutral or protonated species of the inhibitors as shown in the Table . It is noted that the experimental conditions used by Torrisi [START_REF] Torrisi | Discovery and SAR of novel, potent and selective hexahydrobenzonaphthyridinone inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1)[END_REF] for the PARylation of HeLa cells involved stimulation of DNA damage by hydrogen peroxide.

We have previously investigated PARP inhibitors using similar equations [START_REF] Fong | Predicting PARP inhibitory activity -A novel quantum mechanical based model[END_REF] to those in this study, including an investigation of the IC 50 values for more limited number of hexahydrobenzonaphthyridinones. It was also shown that the molecular docking scores [START_REF] Salmas | In silico investigation of PARP-1 catalytic domains in holo and apo states for the design of high-affinity PARP-1 inhibitors, Enzyme Inhib[END_REF] for a wide range of PARP-1 inhibitors were also well described by these equations. [START_REF] Fong | Predicting PARP inhibitory activity -A novel quantum mechanical based model[END_REF]

Conclusions

A study of the in vitro inhibition of hPARP-1 enzyme and PARylation of HeLa cells by a wide range of PARP inhibitors has been conducted utilizing an established linear free energy methodology comprising the free energy of water desolvation and lipophilicity, dipole moment, molecular volume or electron affinity of the PARP inhibitors. It is shown that the IC 50 in vitro inhibition of the hPARP-1 enzyme is closely related to the EC 50 values for PARylation in HeLa cells. The electron affinity of the inhibitors is strongly related to the EC 50 values, suggesting that inhibitor free radicals are involved in oxidative stress processes and PARylation in HeLa cells.

Materials and Methods

All calculations were carried out using the Gaussian 09 package. Energy optimisations were at the DFT/B3LYP/6-31G(d,p) (6d, 7f) level of theory for all atoms. Selected optimisations at the DFT/B3LYP/6-311 + G(d,p) (6d, 7f) level of theory gave very similar results to those at the lower level. Optimized structures were checked to ensure energy minima were located, with no negative frequencies. Energy calculations were conducted at the DFT/B3LYP/6-311+G(d,p) (6d, 7f) level of theory with optimised geometries in water, using the IEFPCM/SMD solvent model. With the 6-31G* basis set, the SMD model achieves mean unsigned errors of 0.6 -1.0 kcal/mol in the solvation free energies of tested neutrals and mean unsigned errors of 4 kcal/mol on average for ions. [START_REF] Av Marenich | Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions[END_REF] The 6-31G(d,p) basis set has been used to calculate absolute free energies of solvation and compare these data with experimental results for more than 500 neutral and charged compounds. The calculated values were in good agreement with experimental results across a wide range of compounds. [START_REF] Rayne | Accuracy of computational solvation free energies for neutral and ionic compounds: Dependence on level of theory and solvent model[END_REF][START_REF] Rizzo | Estimation of Absolute Free Energies of Hydration Using Continuum Methods: Accuracy of Partial Charge Models and Optimization of Nonpolar Contributions[END_REF] Adding diffuse functions to the 6-31G(d) basis set (ie 6-31 + G(d,p)) had no significant effect on the solvation energies with a difference of less than 1% observed in solvents, which is within the literature error range for the IEFPCM/SMD solvent model. HOMO and LUMO calculations included both delocalized and localized orbitals (NBO).

Electron affinities (AEA) in eV in water were calculated by the SCF difference between the optimised/relaxed neutral and optimised radical species method as previously described. [START_REF] Fong | The extravascular penetration of tirapazamine into tumours: a predictive model of the transport and efficacy of hypoxia specific cytotoxic analogues and the potential use of cucurbiturils to facilitate delivery[END_REF] It has been shown that the B3LYP functional gives accurate electron affinities when tested against a large range of molecules, atoms, ions and radicals with an absolute maximum error of 0.

4 Where R 2

 42 ) to the anion radical forms of the hexahydrobenzonaphthyridinone inhibitors: EC 50 values for 25 hexahydrobenzonaphthyridinone anion radical PARP inhibitors Eq 2 = 0.520, SEE = 1197.8, SE(ΔG desol,CDS ) = 252.25, SE(ΔG lipo,CDS ) = 332.51, SE(DM) = 29.89, SE(EA) = 596.82, F=5.15, Significance F = 0.0055 Or by eliminating the non-significant dependency on DM in eq 2, gives eq 2(a): EC 50 values for 25 hexahydrobenzonaphthyridinone anion radical PARP inhibitors Eq 2(a) 50 = . desol,CDS + &. lipo,CDS + ' . !" -20007.7 Where R 2 = 0.520, SEE = 1167.7, SE(ΔG desol,CDS ) = 245.0, SE(ΔG lipo,CDS ) = 313.1, SE(EA) = 543.5, F=7.21, Significance F = 0.0018

  2 eV.[START_REF] Peverati | The quest for a universal density functional: the accuracy of density functionals across a broad spectrum of databases in chemistry and physics[END_REF][START_REF] Jc Rienstra-Kiracofe | Atomic and molecular electron affinities: Photoelectron experiments and theoretical computations[END_REF][START_REF] Ke Riley | Critical Assessment of the Performance of Density Functional Methods for Several Atomic and Molecular Properties[END_REF] Raw MV values have been normalized by factor of 0.1, and raw EA values normalized by 10.0 to allow direct comparison of coefficients in the equations.It is noted that high computational accuracy for each species in different environments is not the focus of this study, but comparative differences between various species is the aim of the study. The literature values for the EC 50 and IC 50 values used to develop the multiple regression LFER equations have much higher experimental uncertainties than the calculated molecular properties. The statistical analyses include the multiple correlation coefficient R 2 , the F test of significance, standards errors for the estimates (SEE) and each of the variables SE(ΔG desolCDS ), SE(ΔG lipoCDS ), SE(Dipole Moment), SE (Molecular Volume), SE (AEA) as calculated from "t" distribution statistics. Residual analysis was used to identify outliers.

	40	-1.33	-7.22	3.43	196
	40 + e -	-1.33	-6.86	13.35	215	1.375
	40 Ion	-4.85	-7.16	15.71	168
	40 + e -	-4.85	-7.16	26.0	196	1.44
	41	-6.07	-8.50	4.36	213
	41 + e -Table 42	-6.07 -1.84	-8.50 -6.80	10.94 2.71	214 239	1.39
	Hexahydrobenzo-naphthyridinone PARP Inhibitors See Figure 2 42 + e -42 Ion 42 Ion + e -43 43 + e -	ΔG desolvation kcal/mol (CDS) Water -1.84 -5.33 -5.33 -2.16 -2.16	ΔG lipophilicity kcal/mol (CDS) n-Octane -6.80 -7.08 -7.08 -6.47 -6.47	Dipole Moment D Water 14.66 22.42 34.0 2.27 12.00	Molecular Volume 238 210 233 cm 3 /mol 213 Water 214	1.38 Electron Affinity eV Water 1.42 1.32
	4 Parent Ion 4 Ion + e -44 44 + e -	-4.30 -4.3 -5.08 -5.08	-5.08 -5.08 -8.99 -8.99	16.8 16.8 5.84 17.97	219 116 116 219	1.54 1.31
	7 Ion 7 Ion + e -45 Ion 45 Ion + e -	-6.00 -6.00 -4.33 -4.33	-8.35 -8.35 -8.31 -8.31	9.3 22.6 33 50.9	237 194 215 252	1.51 1.39
	8 8 + e -47 47 + e -	-5.22 -5.22 -6.58 -6.58	-8.44 -8.44 -11.38 -11.38	9 8.95 3.89 16.25	253 197 197 314	1.31 1.31
	9 9 + e -48 48 + e -	-5.3 -5.43 -6.30 -6.30	-8.97 -8.97 -8.13 -8.13	9 21 2.98 10.76	259 174 194 186	1.31 1.35
	11 11 + e -49 49 + e -	-5.28 -5.28 -6.52 -6.52	-9.24 -9.24 -7.96 -7.96	9.55 22.3 1.38 11.97	206 238 229 215	1.31 1.37
	15	-4.85	-8.64	10.4	242
	15 + e -	-4.05	-8.64	22.0	247	1.42
	16	-5.54	-8.95	6.6	244
	16 + e -	-5.54	-8.95	21.0	204	1.34
	17	-5.15	-8.85	8.8	255
	17 + e -	-5.15	-8.85	22.0	244	1.37
	18	-5.51	-8.83	9.73	216
	18 + e -	-5.51	-8.83	21.8	214	1.34
	20 Ion	-8.19	-10.72	42.7	302
	20 Ion + e -	-8.19	-10.72	37.5	320	1.78
	22	-5.94	-9.49	9.09	212
	22 + e -	-5.94	-9.49	21.89	264	1.34
	23	-5.97	-9.51	8.78	235
	23 + e -	-5.97	-9.51	21.81	246	1.33
	24	-5.85	-9.39	8.91	243
	24 + e -	-5.85	-9.39	23.8	234	1.33
	29	-7.84	-12.02	9.8	293
	29 + e -	-7.84	-12.02	31.1	341	1.34
	33	-3.98	-7.87	10.1	186
	33 + e -	-3.98	-7.87	21.0	196	1.34
	36	-2.93	-7.47	7.9	192
	36 + e -	-2.93	-7.47	13.6	174	1.34
	39	-4.36	-8.07	9.1	180
	39 + e -	-4.36	-8.07	24.2	175	1.31


Carlos Sánchez

Jean-Paul Vidal

Charles-Philippe Lienemann

Jose-Luis Todolí

Evolution of the metal and metalloid content along the bioethanol production process

Keywords: bioethanol, metals, production process, biomass, ICP-MS

Metal concentration has been determined through inductively coupled plasma mass spectrometry for bioethanol samples, raw materials employed to obtain this biofuel and samples taken from different critical points of the production process. In this way it has been possible to study the evolution of the elemental content all along the bioethanol production process, and to establish the origin of the metals present in the final samples, on the other. Moreover, the steps of the production process where metals were either removed from the biomass or accumulated in the biofuel were identified.

Four different acid sample treatments were compared through the analysis of two biomass certified reference materials (CRM) and. the analytical procedure was validated by means of CRMs analysis. The results revealed that, for the most suitable method (nitric acid microwave digestion), recoveries for the analytes of interest went from 90% to 110%. Furthermore, good short-term and long-term precision and acceptable limits of detection (LOD) were obtained.

Two different production lines were studied and slight differences were detected in terms of concentration of minor elements (Cd, Co, Sb, Pb and V). The raw material was identified as the most important source of metals found in the final samples.

Meanwhile the distillation step caused 1000 to 10000 times decrease in metals concentration in the final bioethanol as compared to the initial biomass.

Introduction.

Recently, biofuels have been considered as an effective alternative energy source due to the health and environmental problems caused by fossil fuels together with the increasing demands for energy and the depletion of petroleum in the near future. [START_REF] Demirbas | Competitive liquid biofuels from biomass[END_REF][START_REF] Köpke | Biochemical production of bioethanol[END_REF][START_REF] Walker | Bioethanol: Science and technology of fuel alcohol[END_REF] Among the large number of biofuels, bioethanol is one of the most promising product, largely because its use can reduce up to 75% the emission of greenhouse gases (GHG), as CO 2 , CH 4 and N 2 O, compared with fossil fuels. [START_REF] Demirbas | Competitive liquid biofuels from biomass[END_REF][START_REF] Köpke | Biochemical production of bioethanol[END_REF][START_REF] Walker | Bioethanol: Science and technology of fuel alcohol[END_REF][START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Monot | The NILE Project -Advances in the Conversion of Lignocellulosic Materials into Ethanol[END_REF] Consequently, its production and consumption have grown exponentially during the last two decades. [START_REF] Lemos | Future of Global Bioethanol : An Appraisal of Results , Risk and Uncertainties[END_REF] Bioethanol can be used in its pure form within modified spark-ignition engines or blended with petrol, this blend is known as ethanol-fuel. One liter of ethanol contains 66% of the energy provided by a liter of petrol but it is used in the blend as a very efficient octane-boosting agent, thereby substituting for toxics or chemical additives such as methyl terc-buthyl ether (MTBE). [START_REF] Demirbas | Competitive liquid biofuels from biomass[END_REF][START_REF] Walker | Bioethanol: Science and technology of fuel alcohol[END_REF][START_REF] Nigam | Production of liquid biofuels from renewable resources[END_REF] Although the synthesis process depends strongly on the raw material, the production of bioethanol generally includes the following steps: (i) preparation of the feedstock to achieve maximum yield of sugar content; (ii) hydrolysis of the feedstock and extraction of the sugars; (iii) production of solutions with high sugar concentrations (syrups); (iv) fermentation to convert sugars into ethanol; (v) distillation, which is one of the most important process in terms of pollutants and water removal; and, (vi) dehydration, that is carried out to obtain the ethanol concentration required (anhydrous ethanol or hydrated ethanol). [START_REF] Köpke | Biochemical production of bioethanol[END_REF][START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF] Several industrial processes have been developed to achieve the bioethanol production, appearing different generations of bioethanol. The first-generation bioethanol is the alcoholic product generated from sugars (sugar cane, sugar beet, etc.), seeds or starch (potato, corn, wheat, etc.) using microorganisms. Generally, the yeasts convert sugars in ethanol by fermentation and, after that, the distillation and the dehydration are carried out. [START_REF] Nigam | Production of liquid biofuels from renewable resources[END_REF] Although the process is simple and efficient due to its relatively ease of converting sugars and starch to ethanol, only a reduced part of the plant is used to obtain bioethanol. This causes the main problem of these kind of processes, related to the fuel-food competition. [START_REF] Demirbas | Competitive liquid biofuels from biomass[END_REF][START_REF] Nigam | Production of liquid biofuels from renewable resources[END_REF][START_REF] Fargione | Land Clearing and the Biofuel Carbon Debt[END_REF] The second-generation bioethanol appears to overcome this problem and its production involves a previous enzymatic hydrolysis of agricultural lignocellulosic biomass employed as raw material, which is non-edible food crop production or whole plants biomass. The main advantages of the second generation are that it avoids the fuel-food competition and the low cost of the raw material, which is wasted in other processes [START_REF] Köpke | Biochemical production of bioethanol[END_REF][START_REF] Nigam | Production of liquid biofuels from renewable resources[END_REF][START_REF] Ho | A mini review on renewable sources for biofuel[END_REF]. However, the equipment required to obtain this type of bioethanol is more sophisticated and the yield reached is lower than that obtained in the first-generation processes [START_REF] Nigam | Production of liquid biofuels from renewable resources[END_REF]. Finally, a third generation of biofuels is being developed. Although the third-generation biofuels are being implemented quickly in the case of biodiesel [START_REF] Patil | Towards sustainable production of biofuels from microalgae[END_REF], this emerging technology is still not widely used for bioethanol production.

At the end of the production process, bioethanol may contain organic as well as inorganic pollutants, among them metals. [START_REF] Habe | Chemical Analysis of Impurities in Diverse Bioethanol Samples[END_REF][START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF][START_REF] Sánchez | Metal and metalloid determination in bioethanol through inductively coupled plasma-optical emission spectroscopy[END_REF][START_REF] Chiche | Synthesis Gas Purification[END_REF] The appearance of these metallic species can affect notably the quality of the product. Some of them, as Pb, Tl, Hg, Cd or As, can cause health problems even at low concentrations. [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Donati | Determination of Cr, Ni, Pb and V in gasoline and ethanol fuel by microwave plasma optical emission spectrometry[END_REF][START_REF] Saint'pierre | The development of a method for the determination of trace elements in fuel alcohol by ETV-ICP-MS using isotope dilution calibration[END_REF] Heavy metals are considered an important risk for the environment. [START_REF] Dias | A new highperformance chelation ion chromatographic system for the direct determination of trace transition metals in fuel ethanol[END_REF][START_REF] Saint'pierre | The development of a method for the determination of trace elements in fuel alcohol by electrothermal vaporization-inductively coupled plasma mass spectrometry using external calibration[END_REF][START_REF] Alves | Determination of cadmium in alcohol fuel using Moringa oleifera seeds as a biosorbent in an on-line system coupled to FAAS[END_REF][START_REF] Tormen | Determination of trace elements in fuel ethanol by ICP-MS using direct sample introduction by a microconcentric nebulizer[END_REF] Finally, other metals, as Fe or Cu, can damage the vehicle engines and modify the stability of the ethanol. [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Santos | Simultaneous determination of Cu and Pb in fuel ethanol by graphite furnace AAS using tungsten permanent modifier with co-injection of Ir[END_REF] It is not easy to establish clearly the origin of metals in bioethanol. Some authors reported that they can appear during fuel transportation and storage [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Donati | Determination of Cr, Ni, Pb and V in gasoline and ethanol fuel by microwave plasma optical emission spectrometry[END_REF][START_REF] Dias | A new highperformance chelation ion chromatographic system for the direct determination of trace transition metals in fuel ethanol[END_REF][START_REF] Santos | Simultaneous determination of Cu and Pb in fuel ethanol by graphite furnace AAS using tungsten permanent modifier with co-injection of Ir[END_REF][START_REF] Roldan | Determination of Cu, Ni, and Zn in fuel ethanol by FAAS after enrichment in column packed with 2-aminothiazole-modified silica gel[END_REF] or may be added to the final bioethanol as additive [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Saint'pierre | The development of a method for the determination of trace elements in fuel alcohol by electrothermal vaporization-inductively coupled plasma mass spectrometry using external calibration[END_REF]. However, other works have suggested that the main source of metals in bioethanol is the raw material [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Santos | Simultaneous determination of Cu and Pb in fuel ethanol by graphite furnace AAS using tungsten permanent modifier with co-injection of Ir[END_REF][START_REF] Vieira | Preconcentration and determination of metal ions from fuel ethanol with a new 2,2'-dipyridylamine bonded silica[END_REF][START_REF] Leijenhorst | Inorganic element transfer from biomass to fast pyrolysis oil: Review and experiments[END_REF][START_REF] Vintila | Metal distribution in the process of lignocellulosic ethanol[END_REF][START_REF] Vassilev | An overview of the chemical composition of biomass[END_REF]. Thus, the metal content depends on the atmospheric pollution and the soil where the raw material has grown. [START_REF] Donati | Determination of Cr, Ni, Pb and V in gasoline and ethanol fuel by microwave plasma optical emission spectrometry[END_REF][START_REF] Vintila | Metal distribution in the process of lignocellulosic ethanol[END_REF] Additional works have reported that bioethanol can also be contaminated during its production process. [START_REF] Dias | A new highperformance chelation ion chromatographic system for the direct determination of trace transition metals in fuel ethanol[END_REF][START_REF] Santos | Simultaneous determination of Cu and Pb in fuel ethanol by graphite furnace AAS using tungsten permanent modifier with co-injection of Ir[END_REF][START_REF] Vieira | Preconcentration and determination of metal ions from fuel ethanol with a new 2,2'-dipyridylamine bonded silica[END_REF] Several works have been focused on the development of analytical methodologies to carry out the determination of metals in both bioethanol [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF][START_REF] Sánchez | Metal and metalloid determination in bioethanol through inductively coupled plasma-optical emission spectroscopy[END_REF] and the raw materials employed to obtain this biofuel [START_REF] Leijenhorst | Inorganic element transfer from biomass to fast pyrolysis oil: Review and experiments[END_REF][START_REF] Vintila | Metal distribution in the process of lignocellulosic ethanol[END_REF][START_REF] Vassilev | An overview of the chemical composition of biomass[END_REF], especially when they have been cultivated in a soil contaminated with heavy metals [START_REF] Vintila | Metal distribution in the process of lignocellulosic ethanol[END_REF]. The main conclusion of these works is that a large number of elements were found, at rather low concentrations being Na, Fe, Ni, Co, Cu and K considered as major elements [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF][START_REF] Sánchez | Metal and metalloid determination in bioethanol through inductively coupled plasma-optical emission spectroscopy[END_REF]. However, the number of papers focused on the monitoring of metals along the bioethanol production process is limited and there exist a lack of knowledge in this regard.

The aim of this work was thus to evaluate the fate of metals initially present in the biomass raw material along real bioethanol production processes. This study would get more insight into the possible sources of contamination during this process as well to establish the origin of metals finally present in bioethanol samples. In order to achieve these goals, an analytical methodology based on samples digestion followed by analysis through ICP-MS was developed and validated to finally apply it to the analysis of thirty-two real samples collected from two different processes.

Experimental.

Reagents and standards.

Nitric acid 65% suprapure grade (Merck, Darmstadt, Germany), hydrochloric acid 30% suprapure grade (Merck, Darmstadt, Germany) and hydrogen peroxide 33% w/v (110 vol.) stabilized (Panreac, Barcelona, Spain) were employed for the digestion of the samples.

Four series of multielement standards were prepared from stock solutions to obtain the calibration curves for the analytes of interest: (i) Merck IV solution (Merck, Darmstadt, Germany); (ii) ICP-200.8-1 solution (High purity standards, Charleston, USA); and, (iii) Ca standards were used to obtain the calibration line for this analyte since the calibration with Merck IV solution was not possible because of the spectral interferences caused by 88 Sr 2+ on 44 Ca + and 86 Sr 2+ on 43 Ca + . These three sets of calibration standards were prepared with a solution containing 5% of nitric acid prepared by proper dilution of nitric acid 65% suprapure with ultrapure water (R > 18.2 MΩ) obtained with a water purification system (Millipore, Texas, USA). The blank for the three calibration lines was the aqueous solution with 5% of nitric acid, whereas procedural blanks were obtained for each digestion. Standards and blanks were filtered with a 0.45 µm PTFE filter pore size (Filabet, Barcelona, Spain).

Bioethanol production process and samples.

Thirty-two samples taken along a sugar and bioethanol production processes implemented in a real factory were analyzed. The process studied in the present work is shown in Figure 1. Initially, the pieces of raw material (beetroot) were cut in slices (samples type A) to improve the sugar extraction during the next step and the liquid generated during the sliced process was collected (samples type B). In the second step, the sugars and other compounds (i.e., metals and organic compounds) were extracted using water at 70-80⁰C generating several kinds of juices (samples type C) and the wet material was mechanically pressed and the juice obtained (samples type D) was mixed with those obtained previously. The juice collected (mixture of samples type B, C and D) was treated with calcium hydroxide at temperatures between 40⁰C and 75⁰C (liming) before the addition of carbon dioxide into the solution (carbonatation). The main purpose of these two steps was to remove the metals extracted from the raw material by means of precipitation of insoluble hydroxides and carbonates. After that, the purified juice 1 was heated. Once water was partially evaporated (samples type E), a new liming at 85⁰C and carbonatation generated a cleaner juice (samples type F). In the next step, this juice was heated to generate a syrup with no more than 30% of water and high concentration of sugars (samples type G). After that, some crystallization processes were proposed to obtain sugars from this solution and the rest of the solution was collected and centrifuged. This new syrup (EP2) contained less than 25% of water (samples type H). EP2 was introduced in the fermentation tank and the yeast contained promoted the fermentation process thus giving rise to ethanol causing a depression in the sugars content. Finally, distillation took place obtaining the final bioethanol (samples type J) and the heaviest residue of the distillation process (i.e., vinasse) was used as fertilizer (samples type I).

The samples, provided by the French company UNGDA, were obtained from two different sugar factories located in the center and the east part of France. The sampling procedure was done at the beginning and at the end of the production campaign.

Samples pre-treatment.

Several pre-treatments involving centrifugation, dilution and filtration were tested for liquid samples. However, they were not successful due to the presence and/or formation of suspended solids in the samples. Furthermore, solid samples required a digestion pre-treatment.

Four different digestion pre-treatments were tested and compared for two CRMs: a conventional digestion at atmospheric pressure in a hot plate and three methods based on microwave digestions using different reagents. For the digestion in a hot plate, 5 mL of nitric acid 65% were added to 0.25 g of sample and the mixture was heated at 90⁰C for 1 hour in open glass vessels. The microwave digestion was carried out by adding three different acid solutions to 0.25 g of sample: (i) 8 mL of nitric acid 65%; (ii) 7 mL of nitric acid 65% and 1 mL of hydrogen peroxide 33%; and, (iii) 6 mL of nitric acid 65% and 2 mL of hydrochloric acid 30%. In all the cases, the temperature program consisted of a heating step from room temperature to 200⁰C at a constant rate during 15 min keeping the final temperature constant for 15 min. After the digestion treatment, the volume of the resulting solution was made up to 25 mL with ultrapure water. All the solutions were filtered with a 0.45 µm PTFE filter pore size (Filabet, Barcelona, Spain) at room temperature. Real samples, in turn, were digested using nitric 65% before performing their analysis.

Instrumentation.

A microwave oven Start D (Milestone, Bergamo, Italy) was employed to carry out the samples digestion under controlled pressure and temperature under conditions described in section 2.3.

An Agilent Technologies 7700x ICP-MS spectrometer (Santa Clara, California, USA) equipped with a high matrix introduction system (HMI) was employed to take the ionic intensities of the analytes shown in Table 1. A Scott spray chamber thermostated at -2⁰C and a micronebulizer Micromist® (Glass Expansion, Melbourne, Australia) were established as the sample introduction system. The operating conditions and the isotopes quantified are gathered in Table 1.

Method validation and samples analysis.

Two biomass certified reference materials (CRM), SRM® 1575a (NIST, Maryland, USA) and DC73349 (NACIS, Beijing, China), were employed for method validation. Besides, fifteen spiked real samples and a quality control containing 100 ppb of Merck IV multielement solution (Merck, Darmstadt, Germany) were inserted in the sequence of samples and randomly analyzed to evaluate both, the recoveries obtained in real matrices and the intraday precision. The long-term accuracy and precision was evaluated by means of the analysis of twelve samples that were digested and analyzed with a time gap of three months.

The samples were digested according to the pre-treatment described in 2.3 and they were analyzed by ICP-MS after dilution at an analyte dependent factor. Every sample was digested and analyzed in triplicate. The bioethanol samples (samples J, Figure 1) were analyzed through ICP-MS according a methodology based on external calibration with ethanol/water standards and a total sample consumption system (high temeperature Torch Integrated Sample Introduction System, hTISIS) [START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF].

3. Results and discussion.

Evaluation of the four sample pre-treatment methods.

The results obtained for the two CRMs analyzed are shown in Table 2 andTable 3. Silicon is present in the CRM SRM1575a at 0.6% and it was not completely digested with any of the approaches studied (Table 3). For this reason, the evolution of Si along the process was not studied. Besides, the use of a conventional digestion under atmospheric pressure in open beakers induced systematic errors for several elements. Sodium, boron and barium concentrations were overestimated in CRM DC73349 (Table 2) when the conventional digestion was employed. This could be originated by pollution from the glass containers as an ionic exchange equilibrium was produced between the sample and the surface of the borosilicate glass. [START_REF] Douglas | Reactions of Glasses with Aqueous Solutions[END_REF] Moreover, it should be considered that the contamination suffered by each sample was affected by the specific glass employed, the analytes concentration in the sample and their chemical form. Thus, the found concentrations of Na, B and Ba in CRM SRM 1575a (Table 3), agreed with the certified values because they were present at rather high levels. Other elements, as vanadium, nickel, iron, copper and arsenic provided concentrations statistically lower than the certified values what was assigned to the losses of volatile species and/or non-completed digestion. These results motivated the selection of a microwave based digestion method for pre-treating real samples. Sodium concentrations found in CRM DC73349 after applying the three microwave digestions were slightly higher than the certified level. This could reveal an extra source of contamination. The use of mixed reagents did not provide any further improvement in terms of recovery as compared with the use of only nitric acid. Furthermore, the use of hydrochloric acid or hydrogen peroxide provided higher signals for the procedural blanks than those obtained when only nitric acid was employed. Hence, microwave digestion of 0.25 g of sample with 8 mL of nitric acid was applied as general pre-treatment for all the real samples.

Additionally, carbon ICP-MS signals were monitored for each digestion procedure and they were compared against those obtained for the corresponding procedural blanks.

In the case of microwave treatment methods, carbon ionic intensities were statistically similar to those observed for procedural blanks. Meanwhile, in the case of conventional digestion, dissolved samples provided higher carbon signals than procedural blanks. This fact supported the hypothesis of a non-completed digestion in the case of a conventional acid treatment methodology.

Analytical figures of merit.

The instrumental limits of detection (LODs) were obtained according to the 3 s b /m criterion where s b corresponded to the standard deviation of ten consecutive blank measurements. The LODs for real samples (Table 4) were obtained considering the mass of sample and the final volume of the solution after the digestion procedure.

Inter-day and intra-day precisions were also evaluated. For this purpose, eleven replicates of a QC standard were computed every hour during the sequence, whereas twelve representative samples were selected and the complete analytical procedure was applied to them three months later. In the case of intra-day precision, the relative standard deviation ranged from 2.6% to 6.9% depending on the isotope. For inter-day studies, t paired test was applied for each analyte. The t-values calculated for all the elements quantified were below the tabulated Student-t for a 95% confidence level and 11 freedom degrees (i.e., 2.228). Thus, the methodology based on the samples microwave digestion with nitric acid and the further ICP-MS analysis by using external calibration provided acceptable precision and a satisfying repeatability.

Recovery test.

The recoveries were determined for twelve real matrices. For this purpose, samples were spiked with the analytes of interest and they were analyzed. The added concentration of each analyte was adapted according to the concentration range found in the samples. Additionally, the found concentrations in the non-spiked samples were subtracted to those obtained for the spiked ones. The samples employed in this section were selected based on their representativeness of the complete bioethanol production process. Figure 2 shows the recoveries obtained for each element in a box chart. All the recovery values were included within the 80 to 120% range. Meanwhile, the mean values ranged between 90% and 110%. These results confirmed that matrix effects did not affect the analytical results and the method was accurate (and precise) enough to quantify metals and metalloids in bioethanol related samples.

Analysis of real samples. Metal evolution along the production process.

As mentioned before, two sets of samples coming from different sugar/bioethanol factories were studied in the present work. Although both are based on the same production process (Figure 1), some differences existed between them. In the refinery 1, six points of the process (twelve samples) were analyzed whereas in the process 2 ten points were monitored (twenty samples). The results found for the two treatment plants will be shown separately in order to discern whether variables such as the geographical origin of the raw materials and/or the particular characteristics of the factory (i.e., different critical points) affected the metal content and its evolution throughout the production process. However, it should be mentioned that the samples were collected from a production process that is continuously working and the juices and syrups analyzed were not necessarily coming from the same pieces of raw material as this is not a continuous process. The quality of the raw material might change over time and this might explain some differences. Nonetheless, these samples can be employed to obtain clear conclusions about the possible origin of metals in bioethanol as well as to establish general trend regarding the evolution of these metals along the production process.

Sugar refinery/bioethanol no. 1

Six types of samples were analyzed: raw material (A), juices (C), pressing juices (D), syrup (G), vinasse (I) and bioethanol (J). Each step of the process is described in 2.2.

Figure 3 shows the evolution of the concentration of those elements present above the LOQ from the raw material (beetroot) to vinasse (Figure 1). The elemental concentration in bioethanol samples is not represented as the metals were present at very low concentrations in the final product compared to raw material and intermediate samples. Several interesting conclusions could be drawn from Figure 3.

The major metals present in the raw material were Na, Mg, Ca and K (Figure 3.a and 3.h), were found in the raw material at levels in the order of tens of µg kg -1 . Some of these metals were also typically encountered in bioethanol samples, although they were in concentrations lower than 1 mg kg -1 for major metals and tens of µg kg -1 of trace elements. [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF][START_REF] Sánchez | Metal and metalloid determination in bioethanol through inductively coupled plasma-optical emission spectroscopy[END_REF] As an additional interesting finding, it was clearly noticed that, for Na and K, the concentration in the juice and pressing juice was similar to that found in the raw material. Note that these elements are usually present in biomass under ionic form. [START_REF] Leijenhorst | Inorganic element transfer from biomass to fast pyrolysis oil: Review and experiments[END_REF] Therefore, they can be efficiently extracted in water at 80ºC (See Figure 1).

In contrast, for the remaining elements, the concentration decreased drastically in the extraction step, thus indicating that their extractability in hot water was lower than for Na and K. However, Figure 3 reveals that Mo, Sb, Ba, Co and Mg were more efficiently extracted than Ca, Mn, Zn, Al, Fe, Sr, Ni, Cd and Pb. Although this behavior was shown in beetroot raw material, it could not be extrapolated to any kind of biomass since, for a given element, the extraction yield in hot water would depend on its chemical form. [START_REF] Leijenhorst | Inorganic element transfer from biomass to fast pyrolysis oil: Review and experiments[END_REF] Another interesting phase is the syrup formation and purification (Figure 1). Juice and pressing juice are mixed and limed with Ca(OH) 2 and subsequently carbonated with CO 2 . These two steps are supposed to remove major metals forming insoluble carbonates and hydroxides. The extent of this reaction depends on the solubility product constant, K sp , of each formed salt. Obviously, for a given elemental concentration, the carbonates and hydroxides precipitation with the consequent metal removal, is favored as K sp becomes lower. This constant is in fact very low in the case of divalent and trivalent ions whereas carbonates and hydroxides of monovalent cations show high solubility. In this regard, K and Na concentrations increase around ten times during this step (Figure 3.a). This is because, on the one hand, K and Na carbonates and hydroxides have a high solubility and, on the other hand, water evaporates partially thus leading to a reduction in the total volume. Meanwhile, the rest of the major elements as Ca, Mg, Al, Fe, Ba and Sr are removed from the syrup through insoluble salts precipitation (Figure 3.b, c and e). Additional metals whose solubility product constants were low, such as Co, Ni or Pb, showed a notable rise in their concentration (Figure 3.f and g). This fact could be explained considering that their concentrations in the juice were actually low and, hence, the product of concentrations (e.g., [metal] x [CO 3

2-]) was lower than the solubility constant. As a result, these metals did not form insoluble salts and their growth in concentrations was a consequence of water evaporation.

Finally, distillation is considered to be the most critical step in terms of metals removal.

In the frame of the present study, it was found that the concentration of Na, K, Ca and Mg (major elements) in the obtained bioethanol was in the order of hundreds of µg kg - 1 and below 10 µg kg -1 for the rest of the elements. This meant that the elemental concentrations in the final product decreased by three orders of magnitude as compared to those found in the intermediate products of the process. Although similar elemental concentrations could be expected in syrup and vinasse samples, the concentrations for Na, K, Mn, Zn, Co, Ni, Pb and Mo found in vinasse were in the same order of magnitude to those obtained in the intermediate products and lower than those found in syrup (see Figure 1). A possible explanation to these results would be the precipitation suffered by some elements after distillation.

Focusing on the comparison of the found concentrations of metals in the samples collected at the beginning and at the end of the campaign, it could be concluded that, in general terms, there were differences neither in their evolution along the production process nor in the determined values. Only Pb (Figure 3.g) and Ni (Figure 3.f) concentrations were significantly different as a function of the raw material.

However, in both cases the extracted amount of both metals was virtually the same.

Sugar refinery/bioethanol no. 2

As regards the second bioethanol production refinery, two sets of samples were analyzed: ten samples were collected at the beginning and ten at the end of the production campaign. These samples corresponded to: raw material (A), juice obtained from the sliced raw material (B), extraction juices (C), pressing juice (D), juice heated after first pre-liming and carbonatation (E), juice purified (F), syrup (G), EP2 (H), vinasse (I) and bioethanol (J) (Figure 1). Each step of this process is described in section 2.2.

Although the general trends observed in this process were similar to those shown by the samples coming from refinery 1, samples B, E, F and H were not available in the previous process and they can be helpful for understanding the evolution of metals along the bioethanol production process. Figure 4 shows the evolution of the contents for elements detected at concentrations above 1 mg kg -1 in raw material samples (Na, K, Ca, Mg, Al, Fe, Sr, Ba, Mn and Zn). The corresponding results for elements found at concentrations in the order of tens or hundreds of µg kg -1 are plotted in Figure 5 (Co, Ni, Sb and Mo). Previous reports revealed the presence of all these elements in bioethanol samples. [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF][START_REF] Sánchez | Metal and metalloid determination in bioethanol through inductively coupled plasma-optical emission spectroscopy[END_REF] The concentrations found in the raw material were similar to those obtained in the samples collected from factory number 1. Considering the slicing process, it can be observed that a higher fraction of some metals was extracted at the beginning of the campaign than at its end. This fact can be clearly observed for Fe and Al (Figure 4.c). In the first step of the process, the raw material is cut and separated in two different fractions: slices and the corresponding juice (Figure 1). As a result, metals can partially migrate to the juice. In fact, it was observed that the Fe and Al concentrations in the raw material pieces at the beginning of the campaign were much lower than those obtained at its end whereas the opposite trend was shown for the juice obtained from sliced biomass. This trend was observed in spite to the fact that, along the campaign, the raw material came from different soils and, hence, they might have different elemental composition.

After the raw material was cut, metals were partially extracted with hot water and the solid residue was mechanically pressed. The trends observed in the extraction could be extrapolated from the results obtained for refinery 1. Thus, Na and K were more efficiently extracted than the rest of metals.

In the next step of the bioethanol production process, liming and carbonatation procedures were applied to the blend of three products: juice from slice, juice from extraction and pressing juice (Figure 1). This mix was heated at moderate temperature in order to obtain a solution with 84% of water. Note that in the initial juices, water content went from 86 to 95%. As it was observed in the case of samples from refinery 1, two groups of elements were distinguished: (i) elements removed from the juices through precipitation of insoluble carbonates and hydroxides (Mg, Ca, Fe, Al, Sr, Ba, Mn and Zn); and, (ii) elements that remained dissolved in the solution because either, their carbonates and hydroxides were soluble (Na and K), or they were present in the raw material at actually low concentrations (Sb, Mo, Co and Ni). After the first purification step, a second liming at 85⁰C and carbonatation took place and, as in in the previous purification step, the concentration of Ca, Mg, Fe, Al, Sr, Ba, Zn and Mn decreased (Figure 4) whereas those for Na and K remained unaltered. However, this purification step appeared to cause the partial removal of Co, Ni and Mo (Figure 5).

This was expected, taking into account that the concentration of other elements decreased in the previous purification step and OH -and CO 3 2-ions concentrations in the aqueous solution increased.

Afterwards, the syrup generation and purification was based on a partial evaporation.

For this reason, the concentration of almost all the elements plotted in Figure 4 and Figure 5 notably increased. However, this effect was much more significant when the non-purified syrup (sample G, Figure 1) was converted into EP2 (sample H, Figure 1) by evaporation, centrifugation and crystallization. This was basically caused because, during the crystallization of sugars, metals remained in the solution and the residual syrup (EP2) was enriched in these contaminants. The elemental enrichment factors caused by this evaporation step were included within the 4 to 7 range.

The concentrations found in the final product were in the order of hundreds of µg kg -1 for Na, K, Mg and Ca, and tens of µg kg -1 for the remaining analytes. These results suggested that distillation was an effective process in terms of metals removal since the concentrations found in EP2 were lowered by a factor higher than 1000 in the final bioethanol. In other words, more than 99.9% of the metals present in EP2 remained in the vinasse, which is exploited as fertilizer or animal feeding [START_REF] Christofoletti | Sugarcane vinasse: Environmental implications of its use[END_REF][START_REF] Madejón | Agricultural use of three (sugar-beet) vinasse composts: Effect on crops and chemical properties of a Cambisol soil in the Guadalquivir river valley (SW Spain)[END_REF]. It can be observed that concentrations for Na, K (Figure 4.a), Co, Ni, Sb and Mo (Figure 5) drastically decreased in this point due to precipitation of some metals after distillation.

Anyhow, it is not easy to obtain a clear conclusion in this point as the quality of the raw material is not continuous over the time of the process due to collection of sugar beet obtained from various soils and contaminated with soils particles. .

It is noteworthy that no significant differences were observed between the samples coming from the beginning and the end of the production campaign, except the difference between both set of samples in terms of the fraction of metals extracted by slicing and this derived from the extraction with hot water, which has been previously discussed in this section of the paper.

Finally, when processes for the two evaluated refineries studied were compared, it was concluded that not significant differences were observed between them in terms of evolution of metal concentration along both processes. As regards the elemental concentrations, for major elements, there were not remarkable differences as a function of the raw material. However, the content for trace metals slightly differed for both raw materials. This may be due to differences in the composition of soil where raw materials developed.

It is also interesting to compare the metal contamination reported in the literature for bioethanol [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF][START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF][START_REF] Sánchez | Metal and metalloid determination in bioethanol through inductively coupled plasma-optical emission spectroscopy[END_REF][START_REF] Donati | Determination of Cr, Ni, Pb and V in gasoline and ethanol fuel by microwave plasma optical emission spectrometry[END_REF] and the elements found in the samples analyzed in the present work, collected in these two processes. Most of the elements reported in the literature are initially present in the fermentation step and the transfer of a very small percentage (lower than 0.01%) is observed in the final bioethanol. Sánchez et al. [START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF][START_REF] Sánchez | Metal and metalloid determination in bioethanol through inductively coupled plasma-optical emission spectroscopy[END_REF] reported hundreds of ng mL -1 of Na and K, and tens of ng mL -1 of Ca and Mg in bioethanol samples. In the same work, some trace elements such as Mn, Cu, Fe or Ni were found only in some bioethanol samples in concentrations ranging from 1 to 20 ng mL -1 and other minor elements found in the samples studied in this work, such as Cd, Co, Mo, Pb or Sb, were below the LOQ in all bioethanol samples reported. [START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF][START_REF] Sánchez | Metal and metalloid determination in bioethanol through inductively coupled plasma-optical emission spectroscopy[END_REF] On the other hand, Zn concentrations of hundreds of ng mL -1 were reported in two bioethanol samples [START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF]. However, Zn is not a major element in the samples analyzed in this work. Therefore, bioethanol samples could also suffer metal contamination after their production, during their storage and transport.

Conclusions.

Our initial review [START_REF] Sánchez | Metal and metalloid determination in biodiesel and bioethanol[END_REF] of the literature on the bioethanol contamination and analysis and previous works carried out in the research group [START_REF] Sánchez | Analysis of bioethanol samples through Inductively Coupled Plasma Mass Spectrometry with a total sample consumption system[END_REF][START_REF] Sánchez | Metal and metalloid determination in bioethanol through inductively coupled plasma-optical emission spectroscopy[END_REF] has shown that some elements were systematically observed in the bioethanol produced. In order to verify the potential source of these pollutants, the tracking of metals found in bioethanol samples has been carried out along the production process by means of the use of an analytical method based on ICP-MS after samples microwave digestion, previously validated through the analysis of a biomass CRM and recovery tests. Metals encountered in bioethanol samples have been monitored and they were found in the raw material at concentrations from three to five orders of magnitude higher than those found in the final product. During the bioethanol production process, the liming and carbonatation operations are effective to remove partially metals that can form insoluble hydroxides and carbonates, respectively. However, those elements with high solubility product or present at low concentrations are not removed by this way and their concentrations in the syrups samples increase drastically due to the water evaporation. Finally, during the distillation, metals concentrations decreases by a factor of 1000-10000, showing the effective decontamination of the final bioethanol. It should be mentioned that not important external sources of pollution were discovered along this process, being the most important source of pollution the use of nontreated water that incorporates Ca and Mg. However, this pollution is quantitatively removed during the distillation step.

According to these results, it could be concluded that the main origin of metals in bioethanol commercially available is the raw material, although the steps of the process for removing metals (liming, carbonatation and distillation) seem to be effective, being the distillation the most important and effective operation in terms of metals removal. However, other contamination sources cannot be completely discarded since samples could also be polluted during their storage and transport.

Figure 3 .

 3 Figure 3.b). They were found at concentrations going from 0.1 to 2.2 g kg -1 . Other

Figure 1 .

 1 Figure 1. Scheme of bioethanol production process studied in the present work.

Figure 2 .

 2 Figure 2. Recoveries obtained for twelve spiked samples. Empty squares: average

Figure 3 .

 3 Figure 3. Evolution of metals along bioethanol production process at the beginning and

Figure 4 .

 4 Figure 4. Evolution of major metals along bioethanol production process at the

Figure 5 .

 5 Figure 5. Evolution of minor metals along bioethanol production process at the

Table 1 .

 1 ICP-MS operating conditions.

	Plasma

Table 2 .

 2 Found concentrations for the CRM DC73349 through four different digestion

	methods.*
	Conc. (mg kg -1 )	Certified conc.	MW HNO 3	MW HNO 3 +H 2 O 2 (7:1)	MW HNO 3 +HCl (6:2)	Conventional HNO 3
	Al	580 ± 30	569 ± 12	597 ± 5	559 ± 8	541 ± 6
	B	9.6 ± 0.2	8.2 ± 0.3	3.1 ± 0.4	4.1 ± 0.4	126 ± 2
	Ba	6.0 ± 0.2	5.85 ± 0.17	6.06 ± 0.07	6.66 ± 0.05	43.1 ± 0.4
	Ca	2500 ± 100	2278 ± 38	2367 ± 25	2346 ± 27	2240 ± 23
	Cd	0.233 ± 0.004	0.242 ± 0.010	0.240 ± 0.006	0.252 ± 0.013	0.248 ± 0.011
	Co	0.061 ± 0.002	0.065 ± 0.002	0.060 ± 0.002	0.061 ± 0.003	0.053 ± 0.002
	Cu	2.8 ± 0.2	2.76 ± 0.06	2.79 ± 0.02	2.99± ± 0.04	0.93 ± 0.02
	Fe	46 ± 2	46.6 ± 1.0	46.2 ± 0.4	44 ± 1.0	38.4 ± 0.8
	K	4170 ± 70	4161 ± 60	4284 ± 45	4207 ± 42	4226 ± 50
	Mg	1060 ± 170	1004 ± 16	1015 ± 10	1019 ± 11	994 ± 15
	Mn	488 ± 12	480 ± 11	481 ± 7	482 ± 6	494 ± 4
	N	1.47 ± 0.10	1.63 ± 0.05	1.44 ± 0.03	1.40 ± 0.03	1.27 ± 0.04
	Na	63.0 ± 1.0	84.9 ± 1.3	89.8 ± 1.4	101 ± 5	211 ± 3
	P	1070 ± 80	1049 ± 20	1099 ± 8	1043 ± 10	1063 ± 7
	Se	0.099 ± 0.004	0.089 ± 0.019	0.10 ± 0.03	0.09 ± 0.04	0.14 ± 0.03
	Zn	38 ± 2	38.0 ± 0.5	37.5 ± 0.5	40.9 ± 0.3	33.8 ± 0.5
	*Confidence intervals (N=3 and α=0.05)

Table 3 .

 3 Found concentrations for the CRM SRM 1575a through four digestion method studied.*

	Conc. (mg kg -1 )	Certified conc.	MW HNO 3	MW HNO 3 +H 2 O 2 (7:1)	MW HNO 3 +HCl (6:2)	Conventional HNO 3
	As	1.25 ± 0.15	1.23 ± 0.18	1.23 ± 0.04	1.20 ± 0.03	1.06 ± 0.03
	B	38 ± 6	41 ± 2	37 ± 2	43 ± 2	< LOQ
	Ba	18 ± 2	19.2 ± 0.2	21.0 ± 0.2	15.2 ± 0.2	< LOQ
	Ca	16800 ± 1100	15901 ± 181	16660 ± 202	15771 ± 216	16381 ± 199
	Co	0.41 ± 0.05	0.389 ± 0.003	0.397 ± 0.06	0.401 ± 0.009	0.331 ± 0.003
	Cu	6.6 ± 0.8	5.54 ± 0.04	6.09 ± 0.07	5.38 ± 0.08	3.75 ± 0.07
	Fe	1070 ± 57	941 ± 9	983 ± 10	996 ± 14	629 ± 10
	K	9200 ± 1000	9562 ± 80	9924 ± 108	9482 ± 76	9514 ± 162
	Li	2.6 ± 0.4	2.24 ± 0.18	2.3 ± 0.4	3.5 ± 0.3	2.1 ± 0.3
	Mg	4800 ± 400	4629 ± 48	4787 ± 59	4728 ± 32	4666 ± 78
	Mn	61 ± 5	66 ± 2	68 ± 2	68 ± 2	66 ± 2
	Mo	0.28 ± 0.05	0.271 ± 0.005	0.33 ± 0.02	0.309 ± 0.010	0.264 ± 0.011
	Na	19600 ± 1800	19464 ± 145	20079 ± 274	19920 ± 251	19635 ± 267
	Ni	1.7 ± 0.3	1.39 ± 0.03	1.51 ± 0.05	1.38 ± 0.04	1.21 ± 0.03
	P	1000 ± 40	954 ± 16	1027 ± 16	922 ± 12	990 ± 17
	Pb	47 ± 3	50.5 ± 0.3	50.7 ± 0.8	50.1 ± 0.9	49.0 ± 0.7
	S	7300 ± 600	8662 ± 208	9108 ± 125	8391 ± 132	8655 ± 237
	Sb	0.095 ± 0.014	0.083 ± 0.003	0.132 ± 0.009	0.108 ± 0.004	0.093 ± 0.005
	Se	0.12 ± 0.02	0.18 ± 0.04	0.21 ± 0.03	0.22 ± 0.07	0.16 ± 0.05
	Si	6000 ± 700	381 ± 5	923 ± 7	1506 ± 47	190 ± 4
	Sr	246 ± 16	255 ± 2	255 ± 3	252 ± 2	257 ± 2
	V	2.4 ± 0.4	1.7 ± 0.2	1.8 ± 0.2	1.9 ± 0.2	0.61 ± 0.05
	Zn	55 ± 4	58.2 ± 0.5	62.7 ± 1.0	57.5 ± 0.5	55.3 ± 0.6
	*Confidence intervals (N=3 and α=0.05)

Table 4 .

 4 Limits of detection (in mg kg -1 ) obtained for real samples.

	Analyte	Li	Be	B	Na	Mg	Al	Si	P	S	K
	LOD (mg Kg -1 )	0.16	0.18	2.7	1.0	0.07	0.2	14	0.7	354	2
	Analyte	Ca	V	Mn	Fe	Co	Ni	Cu	Zn	Ga	As
	LOD (µg kg -1 )	1.0	0.003	0.015	0.06	0.008	0.03	0.010	0.04	0.004	0.02
	Analyte	Se	Sr	Mo	Ag	Cd	In	Sb	Ba	Tl	Pb
	LOD (µg kg -1 )	2	0.004	0.002	0.002	0.005	0.002	0.0005	0.009	0.0004	0.002

doi:10.1016/S0167-8809(00)00191-2.

Acknowledgements

The authors wish to acknowledge UNGDA for the samples provided to carry out this work. C. Sánchez thanks the Ministry of Education, Culture and Sports, Spain for the grant FPU 13/01438 and IFPEN for financial support.


L V Abdurakhimov
email: leonid.abdurakhimov@oist.jp

Yu M Bunkov

D Konstantinov

Normal-mode splitting in the coupled system of hybridized nuclear magnons and microwave photons

Keywords: numbers: 71.36.+c, 42.50.Pq, 75.30.Ds, 76.50.+g

In the weak ferromagnetic MnCO3 system, a low-frequency collective spin excitation (magnon) is the hybridized oscillation of nuclear and electron spins coupled through the hyperfine interaction. By using a split-ring resonator, we performed transmission spectroscopy measurements of MnCO3 system and observed, for the first time, avoided crossing between the hybridized nuclear magnon mode and the resonator mode in the NMR-frequency range. The splitting strength is quite large due to the large spin density of 55 Mn, and the cooperativity value C = 0.2 (magnon-photon coupling parameter) is close to the conditions of strong coupling. The results reveal a new class of spin systems, in which the coupling between nuclear spins and photons is mediated by electron spins via the hyperfine interaction, and in which the similar normal-mode splitting of the hybridized nuclear magnon mode and the resonator mode can be observed.

Introduction. When two oscillators are coupled, their otherwise degenerate modes split into two non-crossing hybridized spectrum branches. The phenomenon of avoided crossing is ubiquitous and have been observed in many systems ranging from a classical coupled springmass system [1] to interacting atoms and photons in an optical cavity [2,3], to paramagnetic spin ensembles coupled to microwave cavities [4][5][6][7][8][9]. Systems with strong coupling of microwave photons and spin ensembles are of particular interest since they are proposed to be important building elements of hybrid quantum computers [10,11]. Recently, strong coupling between ferromagnetic electron-spin magnons and microwave photons has been observed in yttrium iron garnet (YIG, Y 3 Fe 5 O 12 ) [12][13][14][15][16][17]. In comparison with the paramagnetic diluted spin ensembles, ferromagnetic materials provide a much higher density of spins which are ordered due to the exchange interaction. Therefore, strong coupling in ferromagnetic systems is characterized by large values of the coupling strength since the coupling constant between a microwave cavity mode and a collective spin excitation (magnon) g m is proportional to the square root of the total number of polarized spins N [18,19], i.e., g m = g 0 √ N , where g 0 is the coupling strength of a single spin to the cavity. Despite the significant progress in studies of strong coupling between microwave cavity mode and electron spin systems, realization of strong coupling between photons and a nuclear spin ensemble is still a challenging problem. Paramagnetic nuclear spins are weakly polarized, and nuclear magnetic moments are typically about 10 3 times smaller than electron magnetic moments. Therefore, the coupling strength between nuclear spins and photons is much weaker in comparison with the electron-spin-photon coupling. To the best of our knowledge, nobody has reported the coupling of a nuclear spin ensemble to a microwave cavity. On the other hand, quantum techniques involving nuclear spins are of great interest due to long nuclear spin decoherence times [20].

In this Letter, we report the first observation of a significant coupling between a nuclear spin ensemble in MnCO 3 system and microwave photons in a cavity. It is manifested as the normal-mode splitting of two coupled oscillators: the hybridized nuclear magnon -homogeneous coupled-spin precession of the nuclei and the electrons (spin wave with k = 0) -and the ac electromagnetic field in the microwave resonator.

The magnetic structure of MnCO 3 crystals was extensively investigated previously [21][22][23][24][25][26][27][28]. The properties of MnCO 3 are summarized hereby briefly. MnCO 3 is a pale-pink transparent crystal with rhombohedral crystal structure. Below the Néel temperature T N ≈ 32.5 K, 55 Mn electron moments form two sublattices that lie in the (111) plane and are canted from perfectly antiparallel antiferromagnetic order due to the Dzyaloshinskii-Moriya (DM) spin-spin interaction (see FIG. 1(a)). Each electron spin sublattice is subjected to an exchange field H E of about 340 kOe [25], an effective magnetic field of DM interaction H DM equal to 4.4 kOe [24][25][26][27][28], and small anisotropy fields which are not relevant to our experiments. Thus, a canting angle α is about one degree, and MnCO 3 is a weak ferromagnet (other examples of weak ferromagnets include α-Fe 2 O 3 [29,30], Y 2 CuO 4 [31], FeBO 3 [32]).

Important feature of MnCO 3 magnetic structure is that 55 Mn nuclear spins (I = 5/2) are coupled to electron spins (S = 5/2) due to the hyperfine interaction [25][26][27](see FIG. 1(a)). An effective magnetic field acting on the nuclear spin system from the electron spin system H n is characterized by the hyperfine coupling constant A, H n = -AM, where M is the magnetization of the electrons of that sublattice on which sites nuclear spins are (color online) (a) Magnetic structure of antiferromagnetic MnCO3 in the (111) plane (y-z plane). Electron magnetic sublattices with magnetizations M1 and M2, respectively, are slightly canted due to the Dzyaloshinskii-Moriya interaction represented by the effective magnetic field HDM . As a result, the non-zero net magnetization M f causes weak ferromagnetism of MnCO3. Nuclear magnetic sublattices with magnetizations m1 and m2, respectively, are coupled to the electron sublattices through the hyperfine interaction fields Hn = -AM i (i = 1, 2). H0 is the external static magnetic field, h1 is the microwave field. (b) Due to the hyperfine coupling, oscillation modes of the electron spin system ωe0 (dashed curve) and the nuclear spin system ωn0 (dashed horizontal line) split into two hybridized branches: the nuclear-ferromagnetic mode ωm (red curve) and the electron-ferromagnetic mode ωe (blue curve). Curves are shown in the two subpanels with different scales along the vertical axis. located. In the magnetically ordered state below the Néel temperature, the magnetization M is near its maximum value, and the field H n reaches tremendous values of about 6 × 10 5 Oe. Similarly, nuclear spins affect electron spins through the hyperfine field H en = -Am, where m is the magnetization of the nuclear sublattice. The hyperfine interaction causes hybridization of the oscillations of electron and nuclear spin systems. In the case of the canted antiferromagnet MnCO 3 , the analysis of the linearized equations of spin motion gives four frequency modes, two for the electron system and two for the nuclear system [22]. One of the electron-frequency modes corresponds to an antiferromagnetic resonance, while the other represents a modified electron-ferromagnetic resonance (EFMR) related to the weak ferromagnetism of MnCO 3 . Correspondingly, there are two hybridized nuclear-frequency modes: a mode of the nuclear spins oscillation coupled to the electron antiferromagnetic mode, and a mode of the nuclear spins oscillation coupled to the electron ferromagnetic mode.

In the experiments reported here, the MnCO 3 system was driven in NMR frequency range by a small magnetic microwave field h 1 that lied in the (111) plane and was perpendicular to the constant external magnetic field H 0 lied at the same plane, i.e., h 1 ⊥ [111] ⊥ H 0 . In this case, only the mode of nuclear spins oscillation coupled to the EFMR mode can be excited at the frequency given by the linear approximation formula [22][23][24][25][26]:

ω 2 m ≡ ω 2 n = ω 2 n0 1 - ω 2 EN ω 2 e . (1)
Here, ω n and ω e = ω 2 e0 + ω 2 EN are the hybridized NMR and EFMR frequencies, ω n0 = γ n H n and ω e0 = γ e H 0 (H 0 + H DM ) are the unmixed NMR and EFMR frequencies, ω EN = γ e √ 2H E Am 0 is the hyperfine interaction parameter, γ n ≈ 2π × 11 MHz/T is the 55 Mn nuclear gyromagnetic ratio [33], γ e = 2π × 28 GHz/T is the electron gyromagnetic ratio, and m 0 = m is the equilibrium nuclear magnetization. The average nuclear magnetization m 0 is determined by the hyperfine field H n as m 0 = χ n H n , with the nuclear magnetic susceptibility χ n changing with nuclear temperature T n according to Curie's law, χ n ∼ T -1 n . Figure 1(b) shows the nuclear-ferromagnetic mode frequency ω m and the electron-ferromagnetic mode frequency ω e calculated from the equations (1) and the related expressions for T = 1.15 K. We used the following values for the corresponding parameters: ω n0 /2π = 640 MHz, H DM = 4.4 kOe, 2H E Am 0 = 5.8 × 10 6 /T n (Oe 2 ×K)≈ 5 × 10 6 Oe 2 (we put T n ≈ T ). The values were taken from [24][25][26][27][28]. Thus, the hybridization of nuclear and electron spins leads to the shift of the NMR frequency -so called "frequency pulling" effect [22] -which has been also observed in CsMnF 3 , FeBO 3 , CoCO 3 , and other materials with the strong hyperfine interaction [27].

It is important to note that an effective microwave field h n acting on the nuclei is considerably larger than the applied microwave field h 1 . Indeed, although the angle of rotation ϕ of the net electron magnetization M f (and, correspondingly, each of magnetizations M 1 and M 2 ) is very small, ϕ ≈ (h 1 /H 0 )(ω 2 e0 /ω 2 e ) [25], each electron sublattice produces a large varying field at the nuclei through the hyperfine field H n . Each nuclear sublattice sees the microwave field

h n = ϕH n = h 1 (H n /H 0 )(ω 2 e0 /ω 2 e
) which is enhanced by the factor η = (H n /H 0 )(ω 2 e0 /ω 2 e ) ≈ 150 relative to the applied microwave field h 1 [23,25].

Experimental setup. In our experiments, we studied the coupling between the hybridized nuclear-electron spin system MnCO 3 and the microwave mode of a split-ring resonator. The experimental setup is shown schematically in FIG. 2. We performed measurements of microwave power transmitted through the resonator as a function of probe microwave frequency and static magnetic field at the temperature T ≈ 1.15 K. The splitring resonator is shown in FIG. 2(b), and, basically, can be approximated by a ring that is split by a gap [34]. The equivalent circuit of the split-ring resonator is the resonant LC circuit determined by the inductance of the single turn coil and the capacitance of the gap. In our experiments, the split-ring resonator was a copper cube with dimensions of 10×10×10 mm 3 , with a centered open-ended bore of about 7 mm in diameter. The volume of the bore cavity V c was about 380 mm 3 . To form the gap of the split-ring resonator, a slit of 0.1 mm was made along the middle line of the top resonator wall, parallelly to the axis of the bore. Thin film of Kapton with thickness of about 0.01 mm was inserted between the cube face with the slit and an additional copper plate with dimensions of 10×10×1 mm 3 , forming a capacitor for adjustment of resonant frequency. In our measurements, the resonant frequency of the split-ring resonator was about ω c /2π ≈ 594 MHz, and Q-factor was about Q c = 100 which is determined by dielectric losses in the Kapton film.

The MnCO 3 single-crystal sample was in the form of a rhombus-shaped plate about 0.7 mm thick, with diagonals of approximately 2.4 mm and 2.7 mm. The [111] crystal axis was perpendicular to the plane of the plate. The mass of the sample was about 8 mg that corresponds to the number of 55 Mn atoms of about N = 4×10 19 . The sample was glued by GE Varnish to the wall inside the cavity of the split-ring resonator.

Normal-mode splitting. Figures 3(a,b) show the microwave transmission |S 12 | 2 = P out /P in as a function of probe microwave frequency f = ω/2π and applied magnetic field H 0 at the probe microwave power of -25 dBm (P in ≈ 3µW) and -10 dBm (P in = 100µW), respectively. Resonances of the system are appeared as yellow or red regions. The horizontal dashed line corresponds to the resonance frequency ω c of the split-ring resonator, while the dashed curve is the frequency ω m of the hybridized nuclear magnon mode calculated by the Eq. ( 1) for T n = T = 1.15 K. When the field approaches the value at which the magnon mode ω m and the microwave cavity mode ω c would cross, a noticeable avoided crossing is observed, indicating normal-mode splitting of the hybridized nuclear magnon mode of the MnCO 3 and the cavity mode.

With the increase of the probe microwave power, the avoided crossing was shifted slightly to the lower mag-netic fields (see FIG. 3(b)). At much higher levels of the probe microwave power (0.3 mW ≤ P in ≤ 10 mW), the avoided crossing was shifted further to low fields, and was significantly distorted as compared with the data presented in FIG. 3 [35]. The shift and the distortion of the splitting shape can be caused by nonlinear spin dynamics. Indeed, the nuclear magnetization m can significantly deviate from its equilibrium direction m 0 at high microwave power, and formulas (1)), derived under the assumption of small oscillations [22], are no longer applicable in this case. Recently, experimental and theoretical studies of nonlinear spin dynamics of MnCO 3 system have been performed, indicating the formation of the magnon Bose-Einstein condensate at high levels of microwave power [36].

We evaluate the coupling strength g m and the magnon and cavity linewidths by fitting the transmission spectrum |S 12 (ω)| 2 with an equation derived from a standard input-output formalism [12,14]:

|S 12 | 2 = √ κ 1 κ 2 i(ω -ω c ) -κ1+κ2+κi 2 + g 2 m i(ω-ωm)-γm 2 2 , (2)
where κ 1 and κ 2 are the external coupling rates to the resonator, κ i is the internal dissipation rate of the resonator, and γ m is the linewidth of the magnon mode. As shown by lines in FIG. 3(c,d), the spectra agree well with the equation ( 2). The parameters obtained by the fitting are g m /2π ≈ 1 MHz, γ m /2π ≈ 3 MHz, and the total resonator linewidth κ/2π = (κ 1 + κ 2 + κ i )/2π ≈ 6 MHz. The cooperativity is calculated to be C = 4g 2 m /κγ m ≈ 0.2 which is close to the conditions of strong coupling regime defined by inequalities g m > κ, γ m and C > 1.

The obtained value of γ m is unexpectedly large. In the MnCO 3 system of coupled nuclear and electron spins at the temperature of T ≈ 1 K, the spin-lattice relaxation time of the hybridized nuclear-frequency mode is expected to be T 1 ≈ 1 ms, while the spin-spin relaxation time should be of the order of T 2 ≈ 10 µs [37]. Therefore, the observed linewidth of the magnon mode γ m /2π = 3 MHz is probably determined by inhomogeneous broadening due to imperfections in the crystal (for example, crystal defects, and crystal inhomogeneities caused by mechanical stresses [25,26]).

Next, we compare the experimentally determined coupling strength g m /2π ≈ 1 MHz with theoretical estimations. The coupling strength between photons and the electron-spin component can be estimated as

g e = (γ e /2) ω c µ 0 /V c √ 2N S × sin(α).
Here, the term sin(α) ≈ (H DM + H 0 )/2H E is added to the standard formula (see, for example, [12][13][14]) to take into account the canting angle α of the electron magnetic moment [29] which determines orientation of electron spins with respect to the microwave field. Inserting the values of the corresponding parameters, we obtain g e /2π ≈ 3 MHz. Similarly, the strength of direct coupling between photons and nuclear spins is

g n = (γ n /2) ω c µ 0 /V c √ 2p n N I × sin(α).
Since the nuclei are paramagnetic, the coupling strength between photons and nuclear spins depends on nuclear spin polarization p n [9]. The net magnetization of the nuclear sublattice containing N spins is m = N γ 2 n 2 I(I + 1)H n /(3k B T n ) [START_REF] Abragam | Principles of Nuclear Magnetism[END_REF], and, hence, spin polarization can be estimated as p n = γ n (I + 1)H n /(3k B T n ). For our experimental conditions, spin polarization p n ≈ 0.5% , and g n /2π ≈ 100 Hz.

By comparing g m with obtained values of g e and g n , we conclude that the direct coupling between nuclear spins and photons is negligible, but the observed normal-mode splitting is mediated by coupling between photons and the electron-spin component of the hybridized magnon mode. Full description of the coupling between electron spins, nuclear spins, and photons is a challenging problem, but some estimations can be done by using the model of the enhanced microwave field h n . As explained in the Introduction, influence of electron spin motion on the nuclei can be taken into account by con-sidering the enhanced microwave field h n acting on the nuclei. The microwave field h n is enhanced by the factor η ≈ H n /H 0 ≈ 150 relative to the applied field h 1 , and is perpendicular to the equilibrium direction of the nuclear magnetization. Hence, the actual coupling strength between nuclear spins and photons should be

G = (γ n /2) ω c µ 0 /V c √ 2p n N I × (H n /H 0 ). Calculated value G/2π ≈ 1 MHz is in very good agreement with the measured value g m .
We also note that g m is independent of the probe microwave power from 3µW to 100µW. This is consistent with results of the works [4,9,12], since the number of photons in the resonator N ω ≈ (Q c P in /ω c )/ ω c 10 13 is much smaller than the number of spins N .

Conclusions.

For the first time, we observed the normal-mode splitting between the hybridized nuclear magnon mode in weak ferromagnetic MnCO 3 and the microwave mode of the split-ring resonator. According to our estimations, the coupling between the paramagnetic nuclear spin ensemble and the microwave cavity mode is mediated by the interaction between photons and the largely-detuned electron ferromagnetic-resonance mode which, in turn, is coupled to nuclear spins via the hy-perfine interaction. The obtained cooperativity value C = 0.2 indicates that our system is close to the conditions of strong coupling. By improving the resonator Q-factor and the quality of crystal, strong coupling can be achieved. The coupling strength can be also increased by cooling the system to millikelvins, and, hence, increasing the nuclear spin polarization. It would be also of great interest to consider possibility of multiple strong coupling under conditions of nuclear electron double resonance [26,[START_REF] Shaltiel | [END_REF] when, in addition to the microwave radiation at the NMR frequency, microwave radiation at the electron FMR frequency is applied (see FIG. 1(b)). We suppose that similar strong coupling phenomena can be realized in other systems with the strong hyperfine interaction (CsMnF 3 , MnF 2 , FeBO 3 , CoCO 3 , and other systems [27]).

The work was supported by an internal grant from Okinawa Institute of Science and Technology (OIST) Graduate University, and JSPS KAKENHI (Grant No. 26400340). Yu.M.B. was supported as Visiting Researcher by Okinawa Institute of Science and Technology (OIST) Graduate University.

FIG. 1 .

 1 FIG. 1.(color online) (a) Magnetic structure of antiferromagnetic MnCO3 in the (111) plane (y-z plane). Electron magnetic sublattices with magnetizations M1 and M2, respectively, are slightly canted due to the Dzyaloshinskii-Moriya interaction represented by the effective magnetic field HDM . As a result, the non-zero net magnetization M f causes weak ferromagnetism of MnCO3. Nuclear magnetic sublattices with magnetizations m1 and m2, respectively, are coupled to the electron sublattices through the hyperfine interaction fields Hn = -AM i (i = 1, 2). H0 is the external static magnetic field, h1 is the microwave field. (b) Due to the hyperfine coupling, oscillation modes of the electron spin system ωe0 (dashed curve) and the nuclear spin system ωn0 (dashed horizontal line) split into two hybridized branches: the nuclear-ferromagnetic mode ωm (red curve) and the electron-ferromagnetic mode ωe (blue curve). Curves are shown in the two subpanels with different scales along the vertical axis.

  FIG. 2. (color online) Experimental technique. (a) Schematic diagram of the experimental apparatus for transmission spectroscopy. (b) The sample of MnCO3 was placed inside the split-ring resonator cavity in a such way that the constant magnetic field H0, the microwave magnetic field h1, and the [111] crystal axis were mutually perpendicular.

FIG. 3 .

 3 FIG. 3. (color online) Transmission spectroscopy of the MnCO3 sample. (a),(b) Transmission spectra as functions of the probe microwave frequency and the static magnetic field for input microwave power of Pin = 3µW and Pin = 100µW, respectively. The horizontal dashed line corresponds to the resonance frequency ωc of the split-ring resonator. The dashed curve is the frequency ωm of the hybridized nuclear magnon mode calculated by the Eq. (1). Normal-mode splitting is observed at approximately H0 = 0.38 T where the magnon mode ωm and the microwave cavity mode ωc would cross otherwise. (c),(d) Cross sections of the spectrum shown on panels (a) and (b), respectively, at static magnetic fields 0.37 T, 0.38 T, and 0.39 T (shown by vertical dashed lines on panels (a),(b)). Dots are experimental data with vertical offset for clarity, and lines are the fitting curves.