Skeletons of Selected Marine Demosponges As Supports for Dyes Adsorption

Home , Chlorophyll f, Hippospongia

Poznan University of Technology Faculty of Chemical Technology Institute of Chemical Technology and Engineering

PhD thesis

Skeletons of selected marine demosponges as supports for dyes adsorption

Małgorzata Norman, M.Sc., Eng.

Supervisor: Professor Teofil Jesionowski

Poznań 2017

Acknowledgement

I would like to express gratitude to many people involved in various stages of development of this doctoral thesis.

First of all, I would like to thank my supervisor Professor Teofil Jesionowski for his scientific support, advice and discussions as well as opportunity to study under his supervision.

I would like to acknowledge Professor Hermann Ehrlich and his team members for all their help during my internship.

I would like to express my gratitude for all my co-authors, colleagues and students from Faculty of Chemical Technology, especially

Filip Ciesielczyk

Jakub Zdarta

Przemysław Bartczak

Sonia Żółtowska-Aksamitowska

Agnieszka Zgoła-Grześkowiak Last, but not least, I would like to thank my Family and Friends, who always supported and encouraged me throughout my life.

Table of contents

Scientific activity ...... 4

List of publications chosen as the basis for the PhD procedure ...... 11

1. Abstract ...... 13

2. Streszczenie ...... 15

3. Introduction ...... 18

3.1. Porifera – state of the art...... 18

3.2. Functionalization of Hippospongia communis skeleton ...... 32

4. Motivation and aim of the work ...... 36

5. Description of the content of publications ...... 37

6. Summary ...... 53

7. List of references ...... 55

Publication 1 ...... 66

Publication 2 ...... 88

Publication 3 ...... 101

Publication 4 ...... 119

Publication 5 ...... 132

Statements of co-authorship ...... 150

Poznan University of Technology

Małgorzata Norman

Scientific activity

Publications:

1. M. Norman, S. ołtowska-Aksamitowska, A.Zgoła Grzekowiak, H. Ehrlich, T. Jesionowski, Iron(III) phthalocyanine supported on a spongin scaffold as an advanced photocatalyst in a highly efficient removal process of halophenols and bisphenol A, Journal of Hazardous Materials 2018, 347, 78-88.

2. J. Zdarta, M. Norman, W. Smułek, D. Moszyński, E. Kaczorek, A.L. Stelling, H. Ehrlich, Teofil Jesionowski, Spongin-based scaffolds from Hippospongia communis Demosponge as an effective support for lipase immobilization, Catalysts 2017, 7(5), 1-20.

3. M. Norman, J. Zdarta, P. Bartczak, A. Piasecki, I. Petrenko, H. Ehrlich, T. Jesionowski, Marine sponge skeleton photosensitized by copper phthalocyanine: A catalyst for Rhodamine B degradation, Open Chemistry 2016, 14, 243-254.

4. M. Norman, P. Bartczak, J. Zdarta, W. Tomala, B. urańska, A. Dobrowolska, A. Piasecki, K. Czaczyk, H. Ehrlich, T. Jesionowski, Sodium copper chlorophyllin immobilization onto Hippospongia communis marine demosponge skeleton and its antibacterial activity, International Journal of Molecular Sciences 2016,17(9), 1564-1580.

5. J. Zdarta, M. Wysokowski, M. Norman, A. Kołodziejczak-Radzimska, D. Moszyński, H. Maciejewski, H. Ehrlich, T. Jesionowski, Candida antarctica ® Lipase B immobilized onto chitin conjugated with POSS compounds: useful tool for rapeseed oil conversion, International Journal of Molecular Sciences 2016,17(9), 1581-1603.

6. M. Norman, P. Bartczak, J. Zdarta, H. Ehrlich, T. Jesionowski, Anthocyanin dye conjugated with Hippospongia communis marine demosponge skeleton and its antiradical activity, Dyes and Pigments 2016, 134, 541-552.

7. P. Bartczak, S. ółtowska, M. Norman, Ł. Klapiszewski, J. Zdarta, A. Komosa, I. Kitowski, F. Ciesielczyk, T. Jesionowski, Saw-sedge Cladium mariscus as a functional low-cost adsorbent for effective removal of 2,4-dichlorophenoxyacetic acid from aqueous systems, Adsorption, 2015, 22(4), 517-529.

8. P. Bartczak, M. Norman, Ł. Klapiszewski, N. Karwańska, M. Kawalec, M. Baczyńska, M. Wysokowski, J. Zdarta, F. Ciesielczyk, T. Jesionowski, Removal of nickel(II) and lead(II) ions from aqueous solution using peat as a low-cost adsorbent: A kinetic and equilibrium study, Arabian Journal of Chemistry 2015, 1-14 (Article in press), doi.org/10.1016/j.arabjc.2015.07.018.

9. Ł. Klapiszewski, T. Rzemieniecki, M. Krawczyk, D. Malina, M. Norman, J. Zdarta, I. Majchrzak, A. Dobrowolska, K. Czaczyk, T. Jesionowski, Kraft

Poznan University of Technology

Małgorzata Norman

lignin/silica–AgNPs as a functional material with antibacterial activity, Colloids and Surfaces B: Biointerfaces 2015, 134, 220-228.

10. J. Zdarta, Ł. Klapiszewski, M. Wysokowski, M. Norman, A. Kołodziejczak- Radzimska, D. Moszyński, H. Ehrlich, H. Maciejewski, A.L. Stelling, T. Jesionowski, Chitin-lignin material as a novel matrix for enzyme immobilization, Marine Drugs 2015, 13, 2424-2446.

11. M. Norman, P. Bartczak, J. Zdarta, W. Tylus, T. Szatkowski, A.L. Stelling, H. Ehrlich, T. Jesionowski, Adsorption of C.I. Natural Red 4 onto spongin skeleton of marine demosponge, Materials 2014, 8, 196-216.

12. J. Zdarta, A. Kołodziejczak-Radzimska, K. Siwińska-Stefańska, K. Szwarc-Rzepka, T. Szatkowski, M. Norman, Ł. Klapiszewski, P. Bartczak, E. Kaczorek, T. Jesionowski, Immobilization of Amano Lipase A onto silica surface: process characterization and kinetic studies, Open Chemistry 2013, 13, 138-148.

13. J. Rakowska, K. Radwan, Z. losorz, B. Porycka, M. Norman, Selection of surfactants on the basis of foam and emulsion properties to obtain the fire fighting foam and the degreasing agent, Tenside Surfactants Detergents 2014, 51, 215-219.

14. M. Nowacka, Ł. Klapiszewski, M. Norman, T. Jesionowski, Dispersive evaluation and surface chemistry of advanced, multifunctional silica/lignin hybrid biomaterials, Central European Journal of Chemistry 2013, 11(11), 1860-1873.

Conference contributions:

1. D. Hernes, M. Norman, A. Zgoła-Grzekowiak, T. Jesionowski, Układ ftalocyjanina żelaza – szkielt gąbki morskiej jako katalizator rozkładu bisfenolu A, II Wielkopolskie Seminarium Chemii Bioorganicznej, Organicznej i Biomateriałów BioOrg, Poznań 02.12.2017 (poster)

2. E. Weidner, M. Norman, A. Zgoła-Grzekowiak, T. Jesionowski, Fotokatalityczna degradacja zanieczyszczeń fenolowych z wykorzystaniem układu hybrydowego szkielet gąbki morskiej – sulfonowana ftalocyjanina żelaza(III), II Wielkopolskie Seminarium Chemii Bioorganicznej, Organicznej i Biomateriałów BioOrg, Poznań 02.12.2017 (poster)

3. M. Norman, D. Hernes, A. Zgoła-Grzekowiak, T. Jesionowski, Usuwanie zanieczyszczeń organicznych w procesie fotodegradacji wspomaganym adsorpcją i utlenianiem, X Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe, nowe wyzwania i moliwoci”, Lublin 21-23.06.2017 (oral presentation)

4. M. Norman, E. Weidner, A. Zgoła-Grzekowiak, T. Jesionowski, Fotokatalityczny rozkład fenolu i jego pochodnych, X Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe, nowe wyzwania i moliwoci”, Lublin 21-23.06.2017 (poster)

Poznan University of Technology

Małgorzata Norman

5. T. Jesionowski, M. Norman, H. Ehrlich, Marine sponge skeleton as a suport for natural dye adsorption, Fifth International Conference on Multifunctional, Hybrid and Nanomaterials, Lisbon 06-10.03.2017 (poster)

6. M. Norman, A. Zgoła-Grzekowiak, T. Jesionowski, Wykorzystanie HPLC jako efektywnej techniki do oznaczania bisfenolu A po procesie jego fotokatalitycznego rozkładu, 5 Konferencja Naukowa „Monitoring i analiza wody. Chromatograficzne metody oznaczania substancji o charakterze jonowym”, Łysomice 02-04.04.2017 (poster)

7. M. Norman, T. Jesionowski, Marine sponge skeleton as a support for dye adsorption, XI Szkoła Letnia dla Doktorantów oraz Młodych Pracowników Nauki Zjawiska Międzyfazowe w Teorii i Praktyce, Sudomie 19-24.06.2016 (oral presentation)

8. T. Szalaty, Ł. Klapiszewski, M. Norman, M. Wrzał, K. Siwińska-Stefańska, T. Jesionowski, Funkcjonalne materiały hybrydowe SiO2-lignosulfonian magnezu otrzymywane z użyciem różnych typów krzemionek, X Konferencja Technologie Bezodpadowe i Zagospodarowanie Odpadów w Przemyle i Rolnictwie, Międzyzdroje 14-17.06.2016 (poster)

9. M. Norman, J. Zdarta, P. Bartczak, T. Jesionowski, H. Ehrlich, Wykorzystanie spektroskopii UV-Vis do oceny wydajności degradacji barwników syntetycznych, IX Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe, nowe wyzwania i moliwoci”, Lublin 07-09.06.2016 (oral presentation)

10. M. Norman, J. Zdarta, P. Bartczak, T. Jesionowski, H. Ehrlich, Metody spektroskopowe w analizie układu metaloftalocyjanina-biopolimer, IX Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe, nowe wyzwania i moliwoci”, Lublin 07-09.06.2016 (poster)

11. J. Zdarta, Ł. Klapiszewski, M. Norman, P. Bartczak, W. Smułek, E. Kaczorek, T. Jesionowski, Ocena aktywności i stabilności enzymów immobilizowanych na matrycach krzemionka-lignina i chityna-lignina w oparciu o pomiary spektrofotometryczne, IX Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe, nowe wyzwania i moliwoci”, Lublin 07-09.06.2016 (oral presentation)

12. J. Zdarta, K. Antecka, M. Norman, Ł. Klapiszewski, P. Bartczak, T. Jesionowski, Wykorzystanie metod spektroskopowych do charakterystyki układu kompozytowego Fe3O4-lignina, IX Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe, nowe wyzwania i moliwoci”, Lublin 07-09.06.2016 (poster)

13. P. Bartczak, M. Wysokowski, F. Ciesielczyk, J. Zdarta, M. Norman, H. Ehrlich, T. Jesionowski, Didymosphenia geminata jako efektywny adsorbent jonów metali szkodliwych dla środowiska, IX Ogólnopolskie Sympozjum „Nauka i przemysł –

Poznan University of Technology

Małgorzata Norman

metody spektroskopowe, nowe wyzwania i moliwoci”, Lublin 07-09.06.2016 (oral presentation)

14. P. Bartczak, J. Zembrzuska, Ł. Klapiszewski, M. Wysokowski, M. Norman, J. Zdarta, F. Ciesielczyk, T. Jesionowski, Adsorpcja fenolu z układów wodnych z wykorzystaniem sorbentów biopolimerowych, IX Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe, nowe wyzwania i moliwoci”, Lublin 07-09.06.2016 (poster)

15. M. Norman, A. Idczak, A. Radkiewicz, J. Zdarta, P. Bartczak, T. Jesionowski, Proces sorpcji metaloftalocyjanin na adsorbencie pochodzenia naturalnego, I Wielkopolskie Seminarium Chemii Bioorganicznej, Organicznej i Biomateriałów BioOrg, Poznań 05.12.2015 (poster)

16. B. urańska, W. Tomala, M. Norman, K. Czaczyk, A. Dobrowolska, T. Jesionowski, Właściwości antybakteryjne materiału hybrydowego chlorofilina - gąbki morskie, I Wielkopolskie Seminarium Chemii Bioorganicznej, Organicznej i Biomateriałów BioOrg, Poznań 05.12.2015 (poster)

17. J. Zdarta, M. Wysokowski, M. Norman, P. Bartczak, A. Jędrzak, T. Szalaty, T. Jesionowski, Układy chityna-POSS jako efektywne nośniki w procesie immobilizacji enzymów, I Wielkopolskie Seminarium Chemii Bioorganicznej, Organicznej i Biomateriałów BioOrg, Poznań 05.12.2015 (poster)

18. P. Bartczak, A. Chudzińska, M. Wysokowski, M. Norman, J. Zdarta, F. Ciesielczyk, H. Ehrlich, T. Jesionowski, Didymosphenia geminata jako nowatorski adsorbent jonów metali szkodliwych dla środowiska, I Wielkopolskie Seminarium Chemii Bioorganicznej, Organicznej i Biomateriałów BioOrg, Poznań 05.12.2015 (poster)

19. T. Szalaty, Ł. Klapiszewski, M. Norman, J. Zdarta, K. Siwińska-Stefańska, T. Jesionowski, Funkcjonalne materiały hybrydowe SiO2-lignosulfonian - otrzymywanie oraz charakterystyka, I Wielkopolskie Seminarium Chemii Bioorganicznej, Organicznej i Biomateriałów BioOrg, Poznań 05.12.2015 (poster)

20. M. Norman, J. Zdarta, P. Bartczak, H. Ehrlich, T. Jesionowski, Gąbki morskie – materiał pochodzenia naturalnego jako skuteczny adsorbent metaloftalocyjanin, II Poznańskie Sympozjum Młodych Naukowców. Nowe Oblicze Nauk Przyrodniczych, Poznań 14.11.2015 (oral presentation)

21. J. Zdarta, Ł. Klapiszewski, M. Norman, A. Jędrzak, T. Szalaty, A. Gan, K. Antecka, T. Jesionowski, Gąbka roślinna Luffa cylindrica jako efektywny nośnik w procesie immobilizacji lipazy z Aspergillus niger, II Poznańskie Sympozjum Młodych Naukowców. Nowe Oblicze Nauk Przyrodniczych, Poznań 14.11.2015 (oral presentation)

22. P. Bartczak, J. Zembrzuska, W. Czernicka, N. Majcherek, M. Norman, M. Wysokowski, Ł. Klapiszewski, F. Ciesielczyk, T. Jesionowski, Układ

Poznan University of Technology

Małgorzata Norman

hybrydowy chityna-lignina − efektywny adsorbent zanieczyszczeń organicznych oraz nieorganicznych, II Poznańskie Sympozjum Młodych Naukowców. Nowe Oblicze Nauk Przyrodniczych, Poznań 14.11.2015 (poster)

23. J. Piotrowska, A. Zdarta, M. Norman, W. Smułek, E. Kaczorek, Oddziaływanie wybranych fungicydów na bakterie glebowe, II Poznańskie Sympozjum Młodych Naukowców. Nowe Oblicze Nauk Przyrodniczych, Poznań 14.11.2015 (poster)

24. J. Zdarta, M. Norman, M. Wysokowski, T. Jesionowski, Zastosowanie nowatorskich materiałów hybrydowych chityna–POSS w procesie unieruchomienia lipazy, 8. Kongres Technologii Chemicznej, Rzeszów 30.08-04.09.2015 (oral presentation)

25. M. Norman, P. Bartczak, J. Zdarta, H. Ehrlich, T. Jesionowski, Immobilizacja sulfonowanych ftalocyjanin Cu(II) oraz Ni(II) na materiałach pochodzenia naturalnego, 8. Kongres Technologii Chemicznej "Surowce - energia - materiały", Rzeszów 30.08-04.09.2015 (poster)

26. M. Norman, P. Bartczak, J. Zdarta, H. Ehrlich, T. Jesionowski, Adsorption of anthocyanins and carotenoids onto biopolymer of natural origin, 9th International Symposium Effects of Surface Heterogeneity in Adsorption and Catalysis on Solids ISSHAC-9, Wrocław 17-23.07.2015 (poster)

27. P. Bartczak, J. Zembrzuska, M. Norman, K. Kabat, F. Ciesielczyk, T. Jesionowski, Coffee grounds as an effective sorbent of phenol from aqueous systems, Ninth International Symposium Effects of Surface Heterogeneity in Adsorption and Catalysis on Solids ISSHAC-9, Wrocław 17-23.07.2015 (poster)

28. K. Siwińska-Stefańska, M. Norman, , T. Jesionowski, Synthesis and characterization of a novel group of TiO2-ZrO2 hybrid materials, Ninth International Symposium Effects of Surface Heterogeneity in Adsorption and Catalysis on Solids ISSHAC-9, Wrocław 17-23.07.2015 (poster)

29. M. Norman, W. Tomala, B. urańska, P. Bartczak, J. Zdarta, T. Jesionowski, Ocena skuteczności adsorpcji chlorofiliny na powierzchni gąbek morskich za pomocą metod spektroskopowych, VIII Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 09-11.06.2015 (poster)

30. J. Zdarta, A. Kołodziejczak-Radzimska, Ł. Klapiszewski, A. Jędrzak, T. Szalaty, P. Bartczak, M. Norman, T. Jesionowski, Weryfikacja skuteczności immobilizacji lipazy na wybranych nośnikach krzemionkowych z wykorzystaniem metod spektroskopowych, VIII Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 09-11.06.2015 (oral presentation)

31. J. Zdarta, A. Kołodziejczak-Radzimska, I. Skotarczak, K. Smelkowska, M. Norman, P. Bartczak, T. Jesionowski, Ocena aktywności immobilizowanych

Poznan University of Technology

Małgorzata Norman

enzymów w oparciu o pomiary spektrofotometryczne, VIII Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 09-11.06.2015 (poster)

32. P. Bartczak, Ł. Klapiszewski, M. Wysokowski, M. Norman, J. Zdarta, F. Ciesielczyk, T. Jesionowski, Ocena efektywności procesu adsorpcji jonów metali na sorbencie typu chityna-lignina z wykorzystaniem technik spektroskopowych, VIII Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 09-11.06.2015 (oral presentation)

33. P. Bartczak, M. Jankowska, J. Zdarta, M. Norman, Ł. Klapiszewski, F. Ciesielczyk, A. Komasa, T. Jesionowski, Usuwanie jonów metali z układów wodnych z wykorzystaniem adsorbentu pochodzenia naturalnego, VIII Ogólnopolskie Sympozjum „Nauka i przemysł – metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 09-11.06.2015 (poster)

34. M. Norman, P. Bartczak, J. Zdarta, Ł. Klapiszewski, T. Szatkowski, H. Ehrlich, T. Jesionowski, Zastosowanie metod spektroskopowych do oceny wydajności procesu adsorpcji barwników naturalnych na gąbkach morskich, VII Ogólnopolskiego Sympozjum „Nauka i Przemysł – metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 10-12.06.2014 (oral presentation)

35. M. Norman, A. Pawełko, D. Połczyńska, W. Król, A. Pisarek, P. Bartczak, M. Wysokowski, T. Szatkowski, T. Jesionowski, H. Ehrlich, Ocena skuteczności adsorpcji barwników naturalnych na powierzchni komercyjnej chityny przy użyciu metod spektroskopowych, VII Ogólnopolskiego Sympozjum „Nauka i Przemysł - metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 10- 12.06.2014 (poster)

36. J. Zdarta, A. Kołodziejczak-Radzimska, Ł. Klapiszewski, P. Bartczak, M. Norman, T. Jesionowski, Weryfikacja skuteczności immobilizacji lipazy na wybranych nośnikach krzemionkowych z wykorzystaniem metod spektroskopowych, VII Ogólnopolskiego Sympozjum „Nauka i Przemysł - metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 10-12.06.2014 (oral presentation)

37. P. Bartczak, Ł. Klapiszewski, M. Norman, J. Zdarta, T. Jesionowski, Zastosowanie atomowej spektrometrii absorpcyjnej w ocenie skuteczności procesu adsorpcji jonów metali na naturalnym torfie, VII Ogólnopolskiego Sympozjum „Nauka i Przemysł - metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 10-12.06.2014 (oral presentation)

38. Ł. Klapiszewski, P. Bartczak, J. Zdarta, M. Norman, T. Jesionowski, Zastosowanie spektroskopii FTIR i XPS w ocenie efektywności wytwarzania materiałów hybrydowych krzemionka-lignina, VII Ogólnopolskiego Sympozjum „Nauka

Poznan University of Technology

Małgorzata Norman

i Przemysł - metody spektroskopowe w praktyce, nowe wyzwania i moliwoci”, Lublin 10-12.06.2014 (oral presentation)

39. P. Bartczak, D. Bednarek, Ł. Klapiszewski, M. Norman, F. Ciesielczyk, T. Jesionowski, Charakterystyka fizykochemiczna i strukturalna torfu jako potencjalnego adsorbentu jonów kadmu z roztworów wodnych, XXXVIII Międzynarodowe Seminarium Naukowo Techniczne „Chemistry for agriculture”, Karpacz, 01-04.12.2013 (poster)

40. A. Kołodziejczak-Radzimska, J. Zdarta, A. Kunierek, M. Norman, T. Jesionowski, Immobilizacja aminoacylazy na powierzchni SiO2, XXXVIII Międzynarodowe Seminarium Naukowo Techniczne „Chemistry for agriculture”, Karpacz, 01-04.12.2013 (poster)

41. J. Rakowska, K. Radwan, Z. losorz, B. Porycka, M. Norman, Emulsion and foams. Structure of surfactant colloids, The 4th International Scientific Conference Applied Natural Sciences 2013 Novy Smokovec, High Tatras, Slovak Republic, 02- 04.10.2013 (poster)

Research projects 1. Research project (Minister of Science and Higher Education) IUVENTUS PLUS V Nr IP2015 032574 - Innowacyjne materiały hybrydowe na bazie ligniny oraz lignosulfonianów aktywowanych cieczami jonowymi, 09.2016-09.2019 Łukasz Klapiszewski – leader, Małgorzata Norman – principal investigator 2. Research project (Poznan University of Technology, Faculty of Chemical Technology) Rola układu krzemionka-lignina w procesie usuwania zanieczyszczeń środowiskowych oraz immobilizacji enzymów, 04-11.2015 Łukasz Klapiszewski – leader, Małgorzata Norman – principal investigator 3. Research project (Poznan University of Technology, Faculty of Chemical Technology) Adsorpcja wybranych barwników organicznych na trójwymiarowych szkieletach gąbek morskich, 04-11.2016 Marcin Wysokowski – leader, Małgorzata Norman – principal investigator 4. Research project (Poznan University of Technology, Faculty of Chemical Technology) Strukturalne kompozyty na bazie trójwymiarowych sponginowych szkieletów gąbek morskich, 04-11.2017 Marcin Wysokowski – leader, Małgorzata Norman – principal investigator

Poznan University of Technology

Małgorzata Norman

Internship

„Inynier Przyszłoci. Wzmocnienie potencjału dydaktycznego Politechniki Poznańskiej” financed by UE, Interdisciplinary Doctoral Studies „Material Science”, internship in TU Bergakademie Freiberg (1.06-31.08.2015)

Awards and scholarships 1. Scholarship of the Minister of Science and Higher Education for outstanding achievements for PhD students in academic year 2016/2017. 2. Scientific scholarship awarded by the Rector of the Poznań University of Technology for the PhD student (2013/2014, 2014/2015, 2015/2016, 2016/2017). 3. Scholarship awarded in the field of Interdisciplinary Doctoral Studies "Material Science" at the Faculty of Chemical Technology at the Poznań University of Technology for the PhD students (2013/2014, 2014/2015, 2015/2016, 2016/2017).

List of publications chosen as the basis for the PhD procedure According to: Ustawa o stopniach naukowych i tytule naukowym oraz o stopniach i tytule w zakresie sztuki (Dz.U. 2003 Nr 65 poz. 595) - 2. Rozprawa doktorska może mieć formę maszynopisu książki, książki wydanej lub spójnego tematycznie zbioru rozdziałów w książkach wydanych, spójnego tematycznie zbioru artykułów opublikowanych lub przyjętych do druku w czasopismach naukowych, określonych przez ministra właściwego do spraw nauki na podstawie przepisów dotyczących finansowania nauki (...).

MNiSW Individual No. Publications IF points input (%) M. Norman, P. Bartczak, J. Zdarta, W. Tylus, T. Szatkowski, A.L. Stelling, H. Ehrlich, T. Jesionowski, Adsorption of C.I. Natural Red 4 onto spongin skeleton of marine demosponge, 1 Materials 2014, 8, 196-216 1.879 35 55 Małgorzata Norman was responsible for preparing and analyzing dye/biopolymer hybrid material as well as writing the manuscript and discussion with reviewers. M. Norman, P. Bartczak, J. Zdarta, H. Ehrlich, 2 T. Jesionowski, Anthocyanin dye conjugated 4.055 40 70 with Hippospongia communis marine

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demosponge skeleton and its antiradical activity, Dyes and Pigments 2016, 134, 541-552 Małgorzata Norman conceived and designed the experiments, developed the results of spectroscopic, thermal analysis and antioxidant tests, wrote the manuscript and discussed with reviewers. M. Norman, P. Bartczak, J. Zdarta, W. Tomala, B. urańska, A. Dobrowolska, A. Piasecki, K. Czaczyk, H. Ehrlich, T. Jesionowski, Sodium copper chlorophyllin immobilization onto Hippospongia communis marine demosponge skeleton and its antibacterial activity, 3 International Journal of Molecular Sciences 3.257 30 45 2016,17(9), 1564-1580 Małgorzata Norman conceived and designed the adsorption experiments, developed the results, wrote the manuscript and answered the reviewers. M. Norman, J. Zdarta, P. Bartczak, A. Piasecki, I. Petrenko, H. Ehrlich, T. Jesionowski, Marine sponge skeleton photosensitized by copper phthalocyanine: A catalyst for Rhodamine B degradation, Open Chemistry 2016, 14, 243-254 4 1.027 20 60 Małgorzata Norman was author of the idea of the research, conducted the adsorption tests as well as catalytic studies, described results of analysis, wrote whole manuscript and discussed with reviewers. M. Norman, S. Zółtowska-Aksamitowska, A. Zgoła-Grzekowiak, H. Ehrlich, T. Jesionowski, Iron(III) phthalocyanine supported on a spongin scaffold as photocatalyst in an advanced oxidation process of halophenols 5 and bisphenol A, Journal of Hazardous Materials 6.065 45 60 2018, 347, 78-88 Małgorzata Norman conceived and designed the adsorption and catalytic experiments, developed the results of hybrid material analysis, wrote the manuscript and answered the reviewers.

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Małgorzata Norman

1. Abstract

During realization of the presented doctoral thesis, the 3D spongin-based hybrid material was obtained and further characterized in terms of its physicochemical and structural properties. The aim of the work was the adsorption of selected dyes, both natural and synthetic, on the sponginous skeleton isolated from the marine sponges. The results of this study allowed to evaluate the effectiveness of the process depending on the contact time between the adsorbent and adsorbate as well as initial concentration of the dye solution, temperature, pH and the ionic strength. Key parameters that have the greatest influence on the process were pH and the presence of Na+ and Cl- ions. The acidic environment proved to be optimal for the adsorption process because it promotes creation of the interactions between functional groups of spongin and dye molecules. In addition, adsorption kinetics and Langmuir and Freundlich isotherm parameters were determined. A kinetic model that uniquely corresponded with experimental data was a pseudo-second order model. In case of adsorption isotherms, the calculated parameters indicate a complex process mechanism. The products obtained during the research were subjected to comprehensive physicochemical and structural analysis using the available methods and techniques. Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray spectroscopy (EDS), carbon nuclear magnetic resonance (13C CP MAS NMR), elemental and thermal analysis were applied. The studies described in the first work Adsorption of C.I. Natural Red 4 onto the spongin skeleton of marine demosponge (M. Norman, P. Bartczak, J. Zdarta, W. Tylus, T. Szatkowski, A.L. Stelling, H. Ehrlich, T. Jesionowski, Materials 2014, 8, 196-216), concerned the adsorption of anthraquinone dye carmine. Results provided interesting and new information about the spongin and answered the question about the nature of the interaction between the sponginous scaffold and dye. The presence of electrostatic interactions and hydrogen bonds between the adsorbent and the adsorbate has been proved. The obtained hybrid material, depending on dye used, exhibited different properties:

 antioxidant – the paper Anthocyanin dye conjugated with Hippospongia communis marine demosponge skeleton and its antiradical activity (M. Norman, P. Bartczak, J. Zdarta, H. Ehrlich, T. Jesionowski, Dyes and Pigments 2016, 134,

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541-552) describe antiradical activity exhibited by anthocyanin dye-spongin 3D hybrid. These properties were tested by estimating the ability of the dye- biopolymer material to scavenge the 2,2'-diphenyl-1-picrylhydrazyl free radical (DPPH∙). Moreover, the equivalent of Trolox was calculated.  antibacterial – the system chlorophyllin/spongin, described in the work Sodium copper chlorophyllin immobilization onto Hippospongia communis marine demosponge skeleton and its antibacterial activity (M. Norman, P. Bartczak, J. Zdarta, W. Tomala, B. urańska, A. Dobrowolska, A. Piasecki, K. Czaczyk, H. Ehrlich, T. Jesionowski, International Journal of Molecular Sciences 2016,17(9), 1564-1580), was verified for the antibacterial activity. A series of tests against Gram-positive and Gram-negative bacteria strains were made, and effective reduction in the growth of Straphylococcus aureus was proved.  catalytic in the process of decomposition of selected organic pollutants: Rhodamine B (Marine sponge skeleton photosensitized by copper phthalocyanine: A catalyst for Rhodamine B degradation (M. Norman, J. Zdarta, P. Bartczak, A. Piasecki, I. Petrenko, H. Ehrlich, T. Jesionowski, Open Chemistry 2016, 14, 243-254)), and phenol, its halogen derivatives and bisphenol A (Iron(III) phthalocyanine supported on a spongin scaffold as photocatalyst in an advanced oxidation process of halophenols and bisphenol A (M. Norman, S. ółtowska- Aksamitowska, A. Zgoła-Grzekowiak, H. Ehrlich, T. Jesionowski, Journal of Hazardous Materials 2018, 347, 78-88)). In these research papers the adsorption of sulfonated copper(II) and iron(III) phthalocyanine on sponginous scaffold was made and the obtained product served as heterocatalyst. According to the obtained results the synergistic effect during the simultaneous use of a heterogeneous catalyst, ultraviolet radiation and hydrogen peroxide was confirmed. Using conjugated technique: high-performance liquid chromatography and mass spectrometry (HPLC-MS), the efficiency of the process was calculated and the degradation products identified.

The preparation of the spongin/dye systems allowed to combine the functional properties of the dyes with the thermally and mechanically resistant natural carrier, the creation of products with unique physicochemical properties and the possibility of finding interesting application.

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Małgorzata Norman

2. Streszczenie

Celem prac była adsorpcja wybranych barwników, zarówno pochodzenia naturalnego, jak i syntetycznych, na sponginowych szkieletach gąbek morskich wyizolowanych z gatunku Hippospongia communis. W ramach przeprowadzonych badań oceniono skutecznoć procesu w zalenoci od czasu kontaktu adsorbent - adsorbat, początkowego stęenia roztworu barwnika, temperatury, pH układu modelowego oraz siły jonowej. Kluczowymi parametrami, w największym stopniu wpływającymi na przebieg procesu było pH oraz obecnoć jonów Na+ i Cl-. rodowisko kwane okazało się optymalne dla procesu adsorpcji, poniewa sprzyja występowaniu oddziaływań pomiędzy grupami funkcyjnymi szkieletów gąbek oraz cząsteczkami barwnika. Ponadto okrelono parametry kinetyczne procesu adsorpcji oraz wyznaczono izotermy wg modeli Langmuira i Freundlicha. Modelem kinetyki, który jednoznacznie korespondował z danymi eksperymentalnymi okazał się model pseudo-drugiego rzędu. W przypadku izoterm adsorpcji obliczone parametry wskazują na złoony mechanizm procesu. W ramach zrealizowanych badań otrzymano, a następnie wnikliwie scharakteryzowano, pod kątem okrelenia właciwoci fizykochemicznych oraz strukturalnych, materiały hybrydowe barwnik-spongina. Wykonano analizy z zastosowaniem spektroskopii w podczerwieni z transformacją Fouriera (FTIR), spektroskopii Ramana, spektroskopii fotoelektronów wzbudzonych w zakresie promieniowania rentgenowskiego (XPS), mikroanalizy rentgenowskiej (EDS), węglowego magnetycznego rezonansu jądrowego (13C CP MAS NMR), analizy elementarnej oraz termicznej (TD/DTA). Rezultaty opisane w pierwszej pracy Adsorption of C.I. Natural Red 4 onto spongin skeleton of marine demosponge (M. Norman, P. Bartczak, J. Zdarta, W. Tylus, T. Szatkowski, A.L. Stelling, H. Ehrlich, T. Jesionowski, Materials 2014, 8, 196- 216), w której barwnik antrachinonowy – karminę zaadsorbowano na sponginowym szkielecie, dostarczyły interesujących informacji wzbogacających wiedzę na temat samej sponginy oraz pozwoliły odpowiedzieć na pytanie o charakter oddziaływań powstałych pomiędzy sponginowym szkieletem a barwnikiem. W oparciu o otrzymane rezultaty stwierdzono występowanie oddziaływań elektrostatycznych oraz wiązań wodorowych pomiędzy adsorbentem i adsorbatem. Wykorzystanie otrzymanych układów w zakresie działania antyrodnikowego było przedmiotem badań zaprezentowanych w publikacji Anthocyanin dye conjugated with

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Małgorzata Norman

Hippospongia communis marine demosponge skeleton and its antiradical activity (M. Norman, P. Bartczak, J. Zdarta, H. Ehrlich, T. Jesionowski, Dyes and Pigments 2016, 134, 541-552). Udowodniono skuteczną aktywnoć przeciwutleniającą układów barwnik antocyjaninowy - spongina, którą oznaczono za pomocą metody redukcji rodnika DPPH (redukcja rodnika 1,1-difenylo-2-pikrylohydrazylu) oraz obliczono równowanik Troloxu. Układy chlorofilina - szkielet gąbki morskiej, opisane w pracy Sodium copper chlorophyllin immobilization onto Hippospongia communis marine demosponge skeleton and its antibacterial activity (M. Norman, P. Bartczak, J. Zdarta, W. Tomala, B. urańska, A. Dobrowolska, A. Piasecki, K. Czaczyk, H. Ehrlich, T. Jesionowski, International Journal of Molecular Sciences 2016,17(9), 1564-1580) zweryfikowano pod kątem aktywnoci antybakteryjnej. Wykonano szereg testów wobec szczepów bakterii Gram-dodatnich oraz Gram-ujemnych, na podstawie których udowodniono działanie ograniczające wzrost bakterii Straphylococcus aureus przez otrzymany produkt. Zweryfikowanie działania katalitycznego w procesie rozkładu zanieczyszczeń organicznych: Rodaminy B (Marine sponge skeleton photosensitized by copper phthalocyanine: A catalyst for Rhodamine B degradation (M. Norman, J. Zdarta, P. Bartczak, A. Piasecki, I. Petrenko, H. Ehrlich, T. Jesionowski, Open Chemistry 2016, 14, 243-254)) oraz fenolu, jego halogenopochodnych jak i bisfenolu A (Iron(III) phthalocyanine supported on a spongin scaffold as an advanced photocatalyst in a highly efficient removal process of halophenols and bisphenol A, (M. Norman, S. ółtowska-Aksamitowska, A. Zgoła-Grzekowiak, H. Ehrlich, T. Jesionowski, Journal of Hazardous Materials 2018, 347, 78-88)) stanowiło cel badań zaprezentowanych w powyszych publikacjach. W pracach tych zaprezentowano wyniki badań dotyczące adsorpcji sulfonowanej ftalocyjaniny miedzi(II) oraz elaza(III) na odpowiednio spreparowanych proteinowych szkieletach gąbek morskich, które to układy pełniły rolę katalizatora w procesie degradacji wymienionych substancji szkodliwych dla rodowiska. Na podstawie zrealizowanych badań stwierdzono efekt synergii w czasie jednoczesnego działania otrzymanego układu heterogenicznego, promieniowania ultrafioletowego oraz nadtlenku wodoru. Wykorzystując wysokosprawną chromatografię cieczową sprzęoną ze spektrometrem mas (HPLC/MS) obliczono wydajnoć procesu oraz zidentyfikowano produkty rozkładu.

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Małgorzata Norman

Otrzymanie trójwymiarowych układów spongina - barwnik pozwoliło na połączenie funkcjonalnych właciwoci barwników z trwałym termicznie i mechanicznie nonikiem pochodzenia naturalnego, stworzenie produktów odznaczających się unikatowymi właciwociami fizykochemicznymi i moliwocią znalezienia ciekawych walorów uytkowych.

Poznan University of Technology

Małgorzata Norman

3. Introduction

3.1. Porifera – state of the art

Sponges, belonging to phylum Porifera, are the phylogenetically oldest animals, their evolution is dating back to 600 million years ago. Currently (2017), there are 8 848 valid species described in the The World Porifera Database [1], which could be found in marine and freshwater habitats. These aquatic animals are currently described in 4 classes: Demospongiae, Calcarea, Hexactinellida and Homoscleromorpha, essentially based on morphological data, molecular and genetic analyses. Sponges, regardless of the class to which they belong, secrete mineral or organic structures that give them a variety of three-dimensional shapes, which minimizes the metabolic cost of water exchange. The skeleton may also be supplemented by exogenous materials, such as sand grains. Most Demospongiae and Hexactinellida produce silica-made skeletons consisting of individualized elements (spicules) of lengths ranging from micrometers to centimeters, which can subsequently fuse or interlock with each other [2]. The high diversity of spicule shapes and sizes in both fossil and living sponges has been repeatedly reported and has received particular attention in taxonomic and cladistic studies. The mineralized spicules of Calcarea class are made of calcium carbonate. Thus, skeletal formations of sponges are examples of natural rigid glass-based or CaCO3 based composites [3]. Sponges’ skeletons, built from minerals and/or organic matrix, depending on the nature and density of building components, may variously be soft, compressible, fragile or hard in consistency. Sponges come in various shapes and sizes, from flat cushions to elaborate branching or cup-shaped forms, from tiny crusts measured in millimeters, to giant shapes in meters. The shapes of sponges are variable among different species and genera, but also vary to some extent between individuals of the same species in response to environmental factors such as hydrodynamics, light and turbidity [4]. The structure of their bodies is very simple and distinguish against other multicellular animals, especially in terms of the lack of organs, tissues and symmetry of the body. They are built form specialized cells for a variety of life functions. The sponge body is made up of two layers of cells: the outer pinacoderm (made of cells called pinacocytes) and the

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Małgorzata Norman inner (choanoderm). Between them there is a mesohyl - an amorphous substance containing cells with various functions. In adult organisms it forms a jelly-like matrix. Pinacocytes forming the outer layer are flat cells capable of shrinking. Porocytes - cells with a water channel, which create a system of ostia responsible for water exchange - are irregularly located between them. The inside of the sponge body is called spongocoel, but it should not be identified with the digestive tract, since spongy digestion takes place inside the cells. Osculum enable water gets out of the body. The above-mentioned elements - pores, ostia and spongocoels together with the osculum form a water system. Three types of Porifera body structure could be distinguish: asconoid, syconoid and leuconoid (Fig. 1.) Asconoid - found in the simplest sponges, is characterized by a single layer of collar cells (chonaocytes) in the spongocoel and straight channels. Choanocytes are cells that line the interior of asconoid, syconoid and leuconoid body type sponges that contain a central flagellum, surrounded by a collar of microvilli. Choanocytes force water and oxygen flow through the spongocoel. Water simultaneously receives carbon dioxide, ammonia and food residue. Syconoid have a tubular body and single osculum, but the body wall, which is thicker and more complex than that of asconoids, contains choanocyte-lined radial canals that empty into the spongocoel. The spongocoel in syconoids is lined with epithelial-type cells rather than flagellated cells as in asconoids. Water enters through a large number of dermal ostia into incurrent canals and then filters through tiny openings into the radial canals. Here food is ingested by the choanocytes, whose flagella force the water through internal pores (apopyles) into the spongocoel. From there it emerges through an osculum. The most developed type of sponge construction is leuconoid, in which chonaocytes are located in spherical chambers forming a network with water channels [5]. It also should be mentioned that sponges have the ability to reproduce the entire organism on the basis of several cells of one kind. Cells connect and then divide, restoring the body.

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Fig. 1. Cross-section of Porifera body structure (a) – asconoid, (b) – syconoid, (c) – leuconoid; 1 – spongocoel, 2 – osculum, 3 – pores, 4 – incurrent canal, 5 – radial canal (choanocyte chamber), red arrow – water flow.

They reproduce in two ways - sexually (gametes occur in mesoglea) or asexually (after fragmentation, by budding and by producing gemmules). Adult forms usually form colonies that can count up to 50,000 individual organisms. Demospongiae is the largest sponge class including about 80% of all living sponges with nearly 7,000 species worldwide, divided into three subclasses: Verongimorpha, Keratosa and Heteroscleromorpha [6]. In Dictyoceratida and Dendroceratida orders, jointly referred to as “keratose demosponges” (also called “horny sponges”), the skeleton does not contain siliceous spicules but only protein (spongin - unique to the phylum Porifera) ﬁbres [7]. Among biomaterials, there are three kinds of organic substances, which skeleton of some sponges can be built of: chitin, spongin and collagen. It was previously shown that chitin is present as a structural component in skeletons of two poriferan classes, Hexactinellida and Demospongiae [8,9]. Taxonomically, spongin is a character of the class Demospongiae. Collagen is the only intercellular organic framework that contribute to approximately 10% of the total organic matter in Demospongiae. In presented doctoral dissertation Hippospongia communis species was used. The taxonomy and distribution of Hippospongia communis are presented in Figs 2 and 3, respectively. Their skeleton is built of spongin ﬁbres exclusively.

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Małgorzata Norman

Fig. 2. Taxonomy of Hippospongia communis.

Fig. 3. Distribution of Hippospongia communis species [10].

Spongin is a biopolymer of still unknown chemical structure, but seems to be a naturally occurring hybrid between collagen and keratin-like proteins. In 1843 Crookewitt first pointed out that the endoskeleton of the common bath sponge is derived from the dermal (horny) layer and called him spongin. Up to now spongin was called also pseudokeratin, neurokeratin, horny protein, collagen-like protein, scleroprotein and spongial multilayered skeleton structures [11]. It is commonly agreed by most authors that

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Małgorzata Norman spongin is formed by epithelial cells - spongioblasts. The basal pinacoderm (basopinacocytes) secrete a mixture of spongin and complex carbohydrates (probably in the form of a fibrillar spongin-polysaccharide complex) that allows the animal to attach to a substrate. The spongin attachment plaques can be seen as the precursor of the sponge holdfast: the protein-carbohydrate-based glue holds the sponge in place [12]. Spongin is also present in the coat of gemmules. Spongin fibres, which may range in thickness from few to several hundred microns, are constituted by densely packed microfibrils arranged within a preferential orientation, usually in concentric layers. The hierarchical, multilevel organization of microfibrils results in spongin, a protein more resistant to enzymatic degradation than collagenous microfibrils themselves. Spongin is known to be resistant to bacterial collagenases, pepsin, trypsin, chymotripsin, pronase, papain, elastase, lysozyme, cellulase, and amylase [13]. Among protein, high sugar content was also typical in number of spongin fibres containing sponges. Glucose, galactose, xylose, mannose, and arabinose were found in conjunction with spongin fibres. Junqua et al. [13] found small amounts of galactosyl-hydroxylysine and much more substantial amounts of glucosylgalactosyl-hydroxylysine in three marine sponges (Ircinia variabilis, Hippospongia communis and Cacospongia scalarist). Fibronectin, a type of glycoprotein, was also found in sponges [14]. On the basis of their amino acid and carbohydrate composition, solubility behavior and presence in the intercellular matrix, they appear to belong to class of structural glycoprotein. Sponge fibronectin-like protein probably play an important role during the reassociation of dissociated sponge as well as during morphogenesis and differentiation of sponges. In some species also lipids were found in their skeletons. What is more important, spongin is defined as a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton and play an important role in formation of extracellular matrix. Genomic and complementary DNA studies showed that spongin (similarly to collagen) contain the classic collagenous Gly-Xaa-Yaa motif where Hydroxyproline (Hyp) occupies any of the positions in the triplet motif, other than Gly (Glycine) position [15]. Non-fibrillar short-chain collagen is likely to be a component of spongin [16]. There is a remote homology between the carboxyl-terminal noncollanenous NC1 domain of spongin short-chain collagens and type IV collagen [17]. The C-terminal, non-collagenous

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Małgorzata Norman domain of individual chains appears duplicated as in the NC1 (non-triple helical) domain of type IV collagen; eight of the nine cysteine residues of this sponge collagen molecule domain are in a similar position as eight of the twelve cysteine residues of the NC1 domain of type IV collagen [15]. Basal membrane with type IV collagen is present in Homoscleromorpha but failed to recover in Demospongiae. Nevertheless, the similarities between spongin, nematode cuticular, and basement membrane type IV collagens could suggest that spongin family possibly reflects two lines of evolution. One line might have been exocollagens (such as spongins) attaching sponges to their substrata (such as worm cuticles). The second might have been internalization of such collagens, leading to the differentiation of basement membrane collagens [18]. Spongin is also analogous to collagen type XIII. The amino acid composition of spongin is similar to vertebrate collagen as determined by infrared absorption spectra, with a high percentage of glycosylated hydroxylysine, aspartic and glutamic acid [19]. Generally, amino acid analysis and morphological studies have shown that sponges possess two different kinds of collagen, structured respectively as microfibrils and fibrils. The microfibrils are involved in the organization of coarse structures, fulfilling a skeletal or protective function. They are restricted to sponges and might be named spongin microfibrils. Basically, they are long filaments about 10 nm in diameter. They can be randomly deposited or regularly arranged, depending on the structure they build. The basal cell layer elaborates a thin sheet of a collagenous material which usually sticks the sponge to its substratum. The unit microfibrils appear then as beaded filaments of about 4 nm in diameter. They can assemble into larger filaments; structures made by two joined microfilaments are rather frequently seen. Spongin microfibrils have to be included in the diverse group of “external collagens”, represented above all among invertebrates (by exoskeleton, cuticles, anchoring devices, etc.) but also in fish [20]. Other study [21] inform that some keratose species possess three characteristic collagens: filamentous, fibrillar (collage type I) and non-fibrillar (type IV), with diverse range of fiber diameter. Studies of Exposito and Garrone [22] revealed that phylum Porifera contains several morphological forms of collagen. The sponge species contain collagen fibrils displaying a typical banding pattern and uniform diameter. In addition, other species possess highly variable forms of collagen aggregates, generally made up of micrifibrils.

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Małgorzata Norman

Spongin matrix has been defined as an exoskeleton and exhibit different morphological aspect among demosponges. Up to now, it is not known if all spongin assemblies are equivalent [17]. Two morphologically distinct forms of spongin fibres, designated spongin “A” and spongin “B,” were demonstrated by Gross et al. to be members of the collagen class [23]. This finding was supported structurally by X-ray diffraction and electron microscopy results, and chemically by their hydroxyproline amid glycine content as well as by the general amino acid pattern. Spongin “A” is a long unbranched fibril of uniform width on the order of 20 nm whereas spongin “B” is a large branched fibre, 10–50 μm in width, composed primarily of bundles of thin unbranched filaments less than 10 nm wide [24]. On the other hand, Garrone has distinguished five types of spongin [25]. First, there is spongin of the spiculated fibres, which is always associated with the endogenous inorganic skeleton of the sponge. This kind of spongin is resistant to diverse bacterial collagenases, pepsin, and mild acid or alkaline hydrolysis. Only a solution of cuprammonium hydroxide appears to be able to attack spongin at room temperature. Second, the spongin fibres which constitute the skeleton of the horny sponges: the abundance and compactness of the spongin and almost complete lack of its own inclusions, which are replaced with foreign particles, testify to the originality of the spongin in this group. Third is the basal spongin, which attaches the animal to the substratum. In sponges with no organized internal skeleton, the more or less continuous layer of external spongin is secreted by the basopinacocytes. In sponges with an organized skeleton, formed either with speculated or spongin fibres, the basal spongin is continuous with the internal spongin. Fourth form, spiculoids are organic elements whose shape, often regular, is strikingly similar to that of inorganic spicules, but extremely flexible and elastic. They are either free or partly joined to the fibres of the skeleton. They are compressible and can be easily torn apart. In its fifth form spongin made a shell of the gemmules [24]. The amino acid composition of spongin, as a proteinaceous material, could vary depending on the sponge species. Nevertheless, among above-mentioned hydroxyproline and glycine glutamic and aspartic acid, arginine, alanine and proline are most popular [26,27]. The results of elemental analysis, energy-dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) of Hippospongia communis spongin [28–30] confirmed the highest content of carbon, oxygen, nitrogen and hydrogen, as expected.

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However, above-mentioned analyses revealed the presence of halogens and other elements as the components of spongin. It has been proved that halogens exist in combination with organic components in demosponges, in form of 3,5-diiodotyrosine and 3-bromotyrosine [24]. Bromine-containing compounds related to tyrosine constitute by far the commonest class of secondary metabolites in Verongida. These compounds are present in the cellular matrix and may also be incorporated into the chitin-based skeletons, where serve as stabilizing and cross-linking substances. Skeletal fibres are thought to play a role in sequestering and accumulating brominated compounds, perhaps as an inactive residues. In Dictioceratida order halogenated tyrosines occur in these sponges together with proteinogenic amino acids. Bromine is often concentrated within keratosan fibres [31,32]. The presence of tyrosine compounds confirmed the presence of aromatic amino acids in spongin, what stays in agreement with (FTIR) and XPS results’(publication 5 [33]), which confirmed the chemical state of carbon characteristic for aromatic compounds. Sulfur is a component of cysteine, an amino acid, which also occurs in the structure of spongin. As it was mentioned, spongin is a protein with similarities to both collagen and keratin. Intra- and intermolecular hydrogen bonds are characteristic for keratin, as well as sulfur- containing cysteine, required for the disulfide bridges that confer additional strength and rigidity by permanent, thermally stable cross-linking. Studies performed and described in presented doctoral dissertation revealed the occurrence of elements at low concentration (Mg, Ca, Al, Si). Presence and concentration of those elements (and others like K, Fe, Mn) stay in agreement with previously published studies and depends on the environment [34]. They are preferentially accumulated either in the skeleton or in the soft tissues depending on the pollution levels of the collection sites. This also allows to consider the use of sponges as biological pollution indicators (biomonitoring). Two kinds of accumulation of elements in sponges could be distinguish: the simple uptake of elements present in environment and particular selection of elements which may be eventually involved in the metabolism of the sponges. In effect, the cleaning procedure of the sponges does not eliminate all the foreign material aggregated in sponge structure. Only the macroscopic materials, like small parts of shell, sand grains etc. may be removed [35]. From practical point of view, the properties and architecture of Hippospongia communis scaffold play a key role and these properties of spongin are consequence of its

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Małgorzata Norman structure. Spongin is insoluble in water and acids. The fibres withstand treatment with 3M HCl and 5% trichloroacetic acid at 90 °C [11]. On the other hand, alkalis can dissolve the spongin material into hydrolysate of amino acids. As it was mentioned above, it is chemically inert and can not be digested by the enzymes. Spongin differs from vertebrate collagens by the presence of unreducible cross-links between the molecules of the tropocollagen elements and aromatic compounds, which provides additional biological stability [19]. Spongin is also thermal stable, the results of studies [36,37] indicate that the degradation of spongin starts at around 150 °C. The thermal stability of spongin stays in agreement with several studies regarding thermal behavior of keratin. Not without a meaning is also a hydration state of collagenous fibrils (presence of water molecules between triple helices), which serve as a mediator in hydrogen bonding between hydroxyl groups of the amino acids, also positively influencing thermal stability. Spongin resembles keratin in its remarkable thermal and chemical stability and resistance to acidic hydrolysis. This protein provide stiffness and strength to the structure in which it occurs. Nonetheless, in modern multicellular animals, spongin gives a sponge its flexibility and support, similar like collagen to a tissue. There is a tremendous diversity of skeletal architectures and fiber constructs within the phylum. The feature, which spongin, collagen and keratin have in common is fibrous structure. Single fibres, composed of microfibers, which are built of amino acid chain, combine into a complex hierarchical network of Hippospongia communis skeleton. The skeleton is three-dimensional, reticular, organized in open-pores cellular structures with multi-junctional regions (Fig. 4).

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Fig. 4. SEM images of Hippospongia communis spongin skeleton at different magnifications.

Stabilization of fibres is affected by the multileveled, hierarchical organization of fibrils and fibres, hydrophobic interactions [13] and the presence of cross-links: (i) between the molecules of the tropocollagen elements and aromatic compounds, (ii) sulfur-sulfur type cross-links between cysteine and cystine residues [19]. Microfibrils have about 10 nm in diameter. Fibres are made of an axial core that may range from a few to about 10-15 μm in thickness, surrounded by helically coiled elementary fibrils which are secreted by the spongioblast cells derived from the mesenchyme. The spongioblast cells arrange themselves in rows and develop a vacuole within which spongin material is collected. Later, spongin secreted by each spongioblast cell fuses with the neighbouring cells to form long fibres. Spongin fibres form a mesh work to provide firmness to the sponge body [38]. The skeletons of Porifera appear to possess several unique and suitable properties: (i) the ability to become hydrated, what is favorable for cell adhesion and (ii) the presence of open interconnected channels created by the ﬁber network makes them an interesting host system, e.g., for cells [19]. Considerable liquid absorption,

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Małgorzata Norman which take place by capillary attraction is due to skeleton’s large internal surface area estimated at between 25 and 34 m2 for a 3 to 4 gram skeleton [39]. Multileveled, cellular and hierarchical structure of spongin skeleton influence their extraordinary mechanical properties at low weight and low density. It is important for effective water pumping and maintains high mechanical strength and endurance necessary against sea and ocean currents as well predators. Moreover, the mechanical performance is crucial from the application point of view. Sponge- or foam-like porous scaffold are widely used in tissue engineering applications, especially for growth of host tissue, bone regrowth or organ vascularization. The porous network simulates extracellular matrix allowing cells to interact effectively with their environment. A detailed study on the mechanical parameters of different species of Demospongiae were conducted by Louden and co-workers [40]. Researchers quantified the physical properties of sponges (density, fibre width and length, water retention efficiency) and their mechanical properties (firmness, compression modulus, compressive strength, tensile strength, elastic limit, elastic strain, modulus of elasticity and modulus of resilience). Sponges are dominant members of many aquatic environments. They interact with wide community in a variety of important relationships, from competing for space with sessile organisms to filtering small suspended particulate matter and transferring energy from the pelagic to the benthic zone. As large biofilters they control microbial populations [41]. From the ecological point of view, individual sponge species can have very specific requirements for substrate quality, food particles, light and current regime. Moreover, sponges are strongly associated with the abiotic environment and therefore they are very sensitive to environmental stress, what makes them useful tools for environmental monitoring [42]. For example sponges have shown accumulation of elements which can be used as a biomarker to assess pollution risks and ecosystem health in the seas and oceans (bioindicators of heavy metals) [43]. Studies of Batista and co-workers [44] evaluates the potential of Hymeniacidon heliophila (Demospongiae class) as bioindicator of polycyclic aromatic hydrocarbons (PAH) contamination. The high rates of filtration - 1 kg of sponges can processes over 24,000 L of water per hour, as well as the ability to ingest particles from 0.2 to 50 mm allows capturing pollutants efficiently both in the dissolved and particulate phases.

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Marine sponges have been used by humans for centuries. The publication of Pronzato and Manconi [45] deals with the history of the exploitation of a natural resource made up of various types of Mediterranean horny sponges. Sponges were used as artistic tool - for painting and decoration. They were widely used in household application for cleaning and hygienic activities. Sponge allowed ancient fishermans to breath during diving and the soldiers served as padding under helmets and armor. Sponges played also a very important role in ancient medicine. The first person who used them for this application was Hippocrates, for which sponges were the primary tool for treating illnesses and health problems. They were used in popular healing baths, as well as for relieving pain. Sponges soaked with hot water were applied to the head, back, hips or legs and were attached with wool or leather. Moreover, small honey-filled sponges were used to treat ear inflammation. They were also an indispensable element in wound dressing - before the bandages were applied, the wound was cleaned and dried with sponges. Sponges also served for digestive system and gynecological diseases treatment. Attached to a string and wrapped in silk were used by ancient Jews, and were historically considered to be the most effective contraceptive. In medieval Arabic surgery sponge soaked with a mixture of hashish, papaver and hyocymine juice, dried under the sun and humidified again when required, were placed at the patient’s nose. In Europe sponge was boiled in a brass vessel with a mixture containing specific proportions of opium, hemlock, and the juices of mandragora, ivy and unripe mulberries until all the liquid was reduced and soaked up in the sponge. The sponge was then applied to the nostrils of the patient. A sponge full of vinegar was usually applied to the nose to wake the patient up again after surgery. Right now the organic chemistry of sponges is also the focus of intense research because they have been known to contain active biomolecules which are of therapeutic and pharmaceutical value. Numerous ecological studies have shown that secondary metabolites produced by sponges often serve defensive purposes to protect them from threats such as predator attacks, microbial infections, biofouling and overgrowth by other sessile organisms [46]. Thousands of sponge-derived bioactive metabolites have been isolated and identified so far [47,48]. Most bioactive compounds from sponges belong to antiinflammatory, antitumor, immuno- or neurosuppressive, antiviral, antimalarial, antibiotic, cytotoxic, cytoprotective, enzyme-inhibitory or antifouling class. One of the example was above-mentioned bromotyrosines. Aeroplysinin-1, a brominated tyrosine metabolite from

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Małgorzata Norman the sponge Verongia aerophoba, has been found to inhibit purified epidermal growth factor receptor protein tyrosine kinase activity, to block proliferation of cancer cell lines, to induce their apoptosis at high nanomolar concentrations and to suppress angiogenesis in vivo [49]. As was previously mentioned, the skeletons of marine sponges are among a group of natural biomaterials that possess elaborate optimized space-filling, three-dimensional architectures. Natural skeletons are highly optimized structures that support and organize functional tissues. They provide important design information for the fabrication of synthetic tissue-engineering scaffolds and may prove efficacious in tissue-engineering strategies [19,50]. The availability, structural diversity, hydrophilic and permeable nature of these natural marine sponges indicate their potential as natural biological scaffolds. Green with co-workers [19] examine the adhesion, spread, growth, differentiation and phenotypic modulation of human osteoprogenitors on the fibre skeletons of a marine sponge. This study also examined the capability of a marine sponge skeleton to act as a delivery vehicle for osteogenic proteins. The results confirm the potential of marine sponge skeletons to deliver bone morphogenic proteins and the advantages of scaffold architecture for bone progenitor cell growth, differentiation and ultimately mineralization. Lin et al. [51] evaluate the usefulness of natural marine sponge collagen as a scaffold for bone tissue engineering. An ideal scaffold for bone tissue engineering must possess suitable biocompatibility, osteoconductive and osteoinductive capacities together with a structure which mimics the trabecular network of bone tissue. Marine sponges display a structure which is similar to the cancellous architecture of bone tissue. The complex canal system within sponges creates a porous environment which is ideal for cellular integration when combined with cells for tissue engineering. The studies of Cunningham and co-workers [52] demonstrates the potential use of marine sponges as precursors in the production of ceramic based tissue engineered bone scaffolds. Three species of Demospongiae: Spongia officinalis, Spongia zimocca and Spongia agaricina were selected for replication. A high solid content of hydroxyapatite was developed, infiltrated into each sponge species and subsequently sintered, producing a scaffold structure that replicated pore architecture and interconnectivity of the precursor sponge. The researchers proved the potential of sponges in repeatable production of hydroxyapatite scaffolds with the necessary characteristics to be used as a viable bone substitute material.

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The perspectives of application (for example in drug delivery systems, cosmetic) of marine sponge collagen was presented in the work of Silva et al. [53]. Ehrlich and Worch [39] demonstrate the examples of sponges as biomaterials – sponges’ spicules of Hexactinellida class are natural glass-based composites with specific mechanical and optical properties. There are also literature reports about surfactant biodegradation by marine sponges [54]. Their potential as an adsorbent for metal uranium [55] as well as dye of natural [28,29,36] and synthetic [30] origin was also demonstrated. Szatkowski and Jesionowski [56] describe the hydrothermal synthesis of materials based on spongin. Authors describe unique structural, mechanical, and thermal properties of chitin extracted from different kind of sponges species and indicate how physicochemical properties may find uses in diverse areas of the material sciences. These new biomaterials have electrical, chemical and material properties that have applications in water purification, medicine, catalysis, and biosensing. The study presented in the work of Zdarta and co-workers described effective immobilization of enzyme - lipase B from Candida antarctica onto 3D spongin-based scaffolds from Hippospongia communis marine demosponge and use of that product for rapeseed oil transesterification [57]. Summarizing, the utilization of materials of natural origin like structural proteins - spongin, has been gaining increasing scientific attention. Key features contributing to the popularity of these biomaterials include biodegradability, ecological safety, low cost, high compatibility with the environment and renewability. Despite the growing interest in sponges, their increasing use is not a threat to them. Spongin-containing marine sponges, including Mediterranean Hippospongia communis, are examples of renewable resources, because are cultivated under marine ranching conditions and represent available and relative low-cost biological materials [58,59]. Literature emphasize the scientific and application potential of marine sponges. Moreover, wide range of sponges’ properties enable further functionalization of selected marine demosponge skeletons as special scaffolds to improve their surface properties and enable their use in various further areas. The functionalization of Hippospongia communis spongin skeleton was made by using dyes. A novel, dye/biopolymer hybrid materials, with designed properties, combine the beneficial features of both constituents.

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3.2. Functionalization of Hippospongia communis skeleton

Functionalization agent used for Hippospongia communis modification described in presented PhD thesis was natural and synthetic dyes with special properties: antioxidant, antibacterial and catalytic properties were used for this purpose.

Dyes derived from nature were the only dyes available to mankind for the coloring until the discovery of the first synthetic dye in XIX century. Natural dyes are derived from plants, animals, microbes or minerals. According to their chemical structure they could be divided into several class, presented in the Fig 5.

Fig. 5. Types of natural dyes with the examples.

Due to the content of the presented publications, only selected, particular dyes will be described in details. Carmine also called C.I. Natural Red 4 or cochineal, is a dark red dye of animal origin [60]. Industrial carmine is obtained by mixing carminic acid with metal salts [61]. The structure of carminic acid is based on anthraquinone with multiple hydroxyl groups,

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Małgorzata Norman a carboxyl group, and a glucose sugar unit side chain. This dye is susceptible to thermal decomposition and photodegradation, but exhibits relatively high chemical and biological stability [62]. It is used mainly for the food, pharmaceuticals and cosmetics coloration, moreover as an indicator in analytical chemistry, in textiles and plastics industry [61], as a dye in microbiology and for the modification of ion exchangers [63]. It is a harmless substance and exhibits many beneficial properties applied in medicine and pharmacy. Carmine can prevent coronary artery disease, plays a role in Alzheimer’s disease treating, exhibits cancer chemopreventive activity and is used in drug delivery products [64].

Other type of dye widespread in nature is anthaocyanin. Anthocyanins are group of plant dyes belonging to the flavonoids, with a polyphenol structure. Different factors affect the color and stability of these compounds, thus their molecules are highly unstable and easily susceptible to degradation under the influence of pH, temperature, ionizing, UV and solar radiation, the presence of copigments, metallic ions, enzymes, oxygen and ascorbic acid [65]. Anthocyanins have electron-donating abilities hence they have great potential for application as sensitizers in photoelectron-chemical dye-sensitized solar cells [66]. In food, cosmetics, and medicines they serve not only as a colorant but also their wide range of bioactive properties and health benefits is exploited. The antioxidant, antibacterial, antiinflammatory, antiviral, antifungal, chemopreventive, anticancer and antimutagenic properties of anthocyanins are well known and described in literature [67,68].

Chlorophyllin, a porphyrin-like structure dye of plant origin is a precursor of chlorophyllin. The stable macrocycle structure (macrocyclic molecule consisting of four pyrrole rings connected by methylene bridges, with a metal ion inside) affect the relative thermal stability of chlorophyllin. This above-mentioned feature as well as water solubility (in contrast to chlorophyll), nontoxicity and numerous of bioactive properties cause the great potential of application for chlorophyllin. Recent studies reveal antioxidant, antimutagenic, and anticarcinogenic properties, because of the ability to bind a planar compounds such as heterocyclic amines, dioxin, aflatoxin and benzo[a]pyrene [69,70]. The antimutagenic activity of chlorophyllin comes also from the scavenging of free radicals and active oxygen species, and suppression of or interference with metabolic activation by a specific cytochrome (P-450) and other metabolizing enzymes. The antibacterial effects of chlorophyllin agains both Gram-positive and Gram-negative strains is fully described

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[71–73]. It also should be mentioned that sodium copper chlorophyllin is used as a dietary supplement, in food, drugs and cosmetics, in textile dye, as an internal deodorizer and as a natural wound healer [74,75]. The promising results obtained for chlorophyllin adsorption (publication 4) inclined to extend the study for other porphyrin-like structure dyes. Phthalocyanines belong to that group. They consist of four isoindole rings connected by azometine bridges. Because of their structure they exhibit unique properties: high redox activity, high molar absorption coefficient (light-absorbing electron donors), thermal stability, oxidant, acid and alkali resistance, as well as high reactivity. These features enable to use phthalocyanines in electronics and sensor application, as a dyes and catalysts or for solar cell production [76]. Metalphthalocyanines exhibit high catalytic activity (in a so-called biomimetic catalytic system) even in ambient conditions, moreover they are relatively easy to synthesize and commercially available [77,78]. However, one limitation of these compounds is that they are inconvenient to use, since they are generally available only as powder, in solution (which hinders their separation from the solution and catalyst recycling) or in the form of thin film [79,80]. Moreover, some molecules are prone to aggregation, which greatly decreases the number of active sites for catalysis [81]. A better strategy for their practical use might be to attach them to a suitable support material. Adsorption of metalphthalocyanines on a solid support is an effective way to remedy the drawbacks of the homogeneous catalyst and enable the creation of a heterogeneous system (possibility of reutilization, increase in the surface area of the catalyst).

For functionalization of Hippospongia communis skeleton and production of spongin/dyes hybrid material the adsorption process was involved. In comparison with other methods this process is fast, facile, takes place in mild conditions and is broadly applied for dyes. Adsorption does not require sophisticated equipment and, what is most important, it is easy to design and implement - there are a lot of parameters, which may be modified to optimize the adsorption conditions. There are two important aspects, which have to be taken into consideration in adsorption process. The first is surface area development of an adsorbent and second adsorbent/adsorbate interactions. As it was mentioned before, the surface area of spongin is estimated at between 25 and 34 m2 for a 3 to 4 grams of skeleton. However in case of Hippospongia communis sponginous skeleton,

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Małgorzata Norman interactions between functional groups of spongin and dyes affect the high efficiency of the adsorption process. For adsorption process crucial is to define the parameters, which influence the adsorption efficiency. Second important aspect is evaluation of adsorption kinetics and modelling of adsorption isotherms. In order to investigate the potential rate-controlling steps, kinetic models have been used to test experimental data. The kinetics of dye adsorption onto adsorbent materials is prerequisite for choosing the best operating conditions. The study of adsorption kinetics illustrates how the solute uptake rate control the residence time of the adsorbate at the solution interface. This rate is most important when designing the adsorption system and this rate can be calculated from kinetic study [82]. In presented PhD thesis the adsorption kinetics parameters were calculated as well as isotherms were presented. Generally, adsorption process involve several steps: (i) transport of adsorbate molecules from the boundary film to the external surface of the sorbent (film diffusion), (ii) transfer of molecules from the surface to the intraparticular active sites and (iii) uptake/interactions of molecules with the active sites of sorbent [83]. The pseudo-first order (PFO) and pseudo-second order (PSO) kinetics model basically include all steps of adsorption such as external film diffusion, adsorption and internal particle diffusion, so they are pseudo-models. It is easily recognized that any of the above steps may be the slowest step determining the overall rate of the interactions and hence the kinetics of the adsorption process. If the step (i) is the slowest, the adsorption will be a transport-limited process (a physical process) and the actual interactions with the solid surface may not be important in determining the adsorption efficiency of the solid. When the step (ii) is the rate determining slowest step, the physical process of diffusion through the liquid film influences the outcome of the process and the efficiency of the solid as an adsorbent can hardly be improved. Only when the step (iii) is the slowest, the adsorption is controlled by a chemical process and the efficiency of the adsorbent can be influenced by suitably controlling interactions [84]. A detailed and thorough description of the adsorption process of above-mentioned dyes onto marine sponge skeleton will be discussed in the chapter 5.

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4. Motivation and aim of the work

The aim of the presented work “Skeletons of selected marine demosponges as supports for dyes adsorption”, schematically presented in Fig. 6, was:

- creating a novel, functional dye/spongin hybrid material with designed properties: antiradical, antibacterial and catalytic,

- functionalization of sponginous skeleton isolated from Hippospongia communis by dye adsorption and utilization of the valuable properties of both constituents,

- evaluation of the influence of several parameters: initial concentration, time, pH, ionic strength, temperature on adsorption efficiency,

- analysis of the physicochemical and morphological properties of the obtained materials.

Moreover there is also a scientific objective: broadening the knowledge about spongin chemistry, characterization and utilization.

Fig. 6. Schematic representation of the objectives of PhD thesis.

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5. Description of the content of publications

The studies described in publications 1-5 are focused on the search and development of a new type of functional material of natural origin with designed properties. Hippospongia communis sponginous skeleton with three-dimensional architecture and presence of reactive functional groups was selected as a new type of support for dyes adsorption. Natural dyes derived from plants and animal sources are used by people for centuries. Recently are widely-studied compounds in modern science and new information about their anti-cancer, anti-inflammatory and antioxidant properties are published [85– 88]. Natural colors have some disadvantages as compared to synthetic ones, as they are more expensive and less stable, susceptible to degradation affected by light, pH, temperature, sulfite, ascorbic acid and enzymes [65]. Natural dyes are renewable and sustainable bioresource products with minimum environmental impact, biodegradable and non-toxic [89]. They find applications in food and textile coloration, as pH indicators, in cosmetics, pharmaceuticals or in dye sensitized solar cell production [90]. However, the most important limitation reported is the low bio-distribution and bioavailability as well as instability. Therefore, adsorption of these natural compounds onto biopolymers support should improve their stability and enable use of their antibacterial and antiradical properties in further applications. Taking the above-mentioned facts into account, the first step of all experimental work, preceded by isolation of the sponginous 3D skeleton, was the adsorption process. Adsorption seems to be versatile process for hybrid material preparation due to its mild conditions, simplicity and limited cost. In presented publications the selection of optimal adsorption parameters in order to obtain product with high content of dye was of particular importance. Fig. 7 shows the effect of different dyes concentration on the amount of the dye adsorbed (qt) on the H. communis spongin scaffold, plotted against time.

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Fig. 7. Adsorption capacity of carmine, anthocyanin, sodium copper chlorophyllin and copper phthalocyanine on sponginous skeleton, as a function of time.

The studies described in publication 1 concerned the adsorption of carmine. It was concluded that several parameters may determine its efficiency. This includes the concentration of the adsorbate solution, the pH and the contact time of the reagents. The higher carmine concentration and duration of adsorption the higher amount of dye was adsorbed onto spongin skeleton. Nevertheless, the crucial parameter was pH: the acidic conditions promotes the adsorption process. A number of studies have found that dependency on pH occurs when interactions between functional groups of adsorbent and adsorbate take place [91]. In publication 2, regarding anthocyanins adsorption, the optimum pH was 4 and both decrease and increase of pH of dye solution caused decrease in adsorption efficiency. This behavior is related to the presence of different forms of anthocyanin depending on the different pH values (Fig. 8).

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Fig. 8. Different forms of anthocyanin, depending on pH and possible interactions of anthocyanin with spongin in different pH (image copyrights – Elsevier) [28].

In publication 2 there is a detailed description of the chemical structures of the various forms of anthocyanin as well as their interaction with spongin. Higher content and longer contact time between adsorbent and adsorbate favors the adsorption process. An analysis was also made of the effect of temperature on the adsorption process of anthocyanin dye. The results show that an increase from 20 to 40 °C is favorable to the adsorption process. An increase in the temperature improve the adsorption efficiency which suggests an endothermic nature of the process. This may result from increase in the mobility, and hence energy of the dye molecules, which exhibit in higher temperature. Moreover, the decrease in the thickness of the boundary layer surrounding the adsorbent causes a decrease in the mass transfer resistance of the adsorbate [92]. The research described in publication 3, in which sodium copper chlorophyllin (SCC) was adsorbed, confirmed the previously obtained results. High dye solution concentration, long duration of adsorption process as well as acidic pH causes the increase in the adsorption efficiency. The another factor which influences the efficiency was presence of additional

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Małgorzata Norman ions, Na+ and Cl-. Generally, for all tested dyes the increase in NaCl concentration causes the increase in adsorption efficiency. This behavior may be caused by several factors. According to the surface chemistry theory, if there is an attractive electrostatic interaction between adsorbate and adsorbent, then an increase in ionic strength should cause a decrease in the adsorption capacity of the adsorbent. The converse is found in case of repulsive interaction; increasing ionic strength then causing an increase in adsorption capacity, especially when the concentration is high. The presence of additional ions from salt in the medium decreases the repulsion between adjacent dye particles, allowing the adsorbed molecules on the surface to be closer to each other. In addition, the electric double layer, which surrounded both adsorbent and adsorbate, is compressed at high ionic strength, resulting in lowering or elimination of the repulsive energy barrier. Moreover van der Waals forces, ion-ion interactions and dipole-dipole interactions increased [93,94]. The realization of the adsorption process of copper phthalocyanine (denoted as CuPC) using Hippospongia communis skeleton was described in publication 4. Analogous dependencies in realization of adsorption process were observed. The obtained experimental data confirmed that the amount of dye adsorbed increased proportionally to the increase of concentration of model solutions, increased amount of NaCl and contact time. The pH of the reaction is one of the major factors which influences adsorption efficiency. Acidic pH promotes adsorption by affecting the interaction between dye and spongin skeleton. CuPC have in their structure functional groups (sulfonic) capable for dissociation. Spongin, as a protein material is built of several amino acids with characteristic amino groups (–NH2), which could be protonated into form of cationic (– + NH3 ). Therefore, according to obtained results, the interaction between oppositely charged functional groups are possible. Table 1 presents the optimal adsorption condition for all tested dye. Table 1. Optimal adsorption condition for tested dyes.

Dye Adsorption conditions carmine pH=3, 60 min anthocyanins pH=4, 40 °C, 0,01 M NaCl, 360 min chlorophyllin pH=5, 0,01 M NaCl, 90 min copper phthalocyanine pH=2, 1 M NaCl, 360 min

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The determination of sorption capacity of spongin was also an important aspect of the studies. The well-known and commonly described Langmuir and Freundlich isotherm models were used for this purpose. The equation of these models are presented in Table 2.

Table 2. Nonlinear form of isotherm models.

Isotherm model Nonlinear form

Langmuir 푞 ∙ 푏 ∙ 퐶 푞 = Freundlich + 푏 ∙ 퐶 푞 = 퐾 ∙ 퐶 where: qe are the amount of dye adsorbed at equilibrium respectively (mg/g), Ce denotes the equilibrium concentration of the dye solution (mg/L), qm is the maximum adsorption capacity, and b is the Langmuir constant (related to the affinity of the binding sites and the bonding energy of adsorption) (L/mg) while Kf (mg/g) and n (related to adsorption intensity) are the Freundlich constants. Analysis of parameters of adsorption isotherms indicated a notable sorption capacity of sponginous scaffold towards the adsorbed dyes (1413.9 mg/g for anthocyanins, 108.56 mg/g for SCC, 83.36 mg/g for CuPC and 18.55 mg/g for carmine) (Table 3). The differences in the amount of adsorbed dyes may be caused by many factors. Attention should be given to the chemical structure of the dye, its molecular weight or the number and reactivity of functional groups. Additionally, it is worth noticing that the discussed adsorption process of carmine was more accurately described using the Langmuir isotherm model, which is confirmed by the R2 correlation coefficient value of 0.995. In case of anthocyanins, chlorophyllin and copper phthalocyanine this parameter was higher for Freundlich model (Table 3). On the other hand, there is no significant difference between correlation coefficient (R2) calculated according to Freundlich and Langmuir model. Isothermal parameters do not point clearly to either the Freundlich or the Langmuir equation, which suggests a combined sorption mechanism. For all studied dyes the obtained values of the n coefficient are higher than 1, therefore adsorption process involves physical interaction between adsorbent and adsorbate. Nevertheless, results of spectral analyses, suggest also chemical interactions what indicate the complex mechanism of adsorption.

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Table 3. Parameters of isotherm models of dye adsorption on sponginous skeleton isolated from Hippospongia communis.

Isotherm model Langmuir Freundlich

Parameters qm b KF R2 R2 n Dye (mg/g) (L/mg) (mg/g)

carmine 0.995 18.55 0.034 0.877 3.61 4.19

anthocyanins 0.863 1413.9 0.001 0.986 1.47 1.78

chlorophyllin 0.992 108.56 0.15 0.995 13.97 2.27

copper phthalocyanine 0.917 83.36 0.03 0.957 14.09 3.46

In order to define the mechanism of interactions at the adsorbent/adsorbate interface, a theoretical description of the process using kinetics models was conducted. Pseudo-first order and pseudo-second order kinetic models were fitted to the experimental data. These models present the correlations between changes in the concentration of adsorbate as a function of time within the adsorption process is continued, until equilibrium is reached. The equations of above-mentioned models are presented in Table 4.

Table 4. Linear form of adsorption kinetic models.

Kinetic model Linear form

pseudo-first order 푘 log푞 − 푞푡 = log푞 − · 푡 pseudo-second order . 푡 = + · 푡 where: qt and qe denotes the amount of adsorbed dye at 푡 time t and at equilibrium 푞 푘 푞 푞 -1 respectively (mg/g), k1 is the rate constant of pseudo-first order adsorption (min ), where -1 k2 (g (mg min) ) is the second order rate constant of adsorption, and t is the contact time (min). The determined kinetic parameters indicate that for all tested dyes the obtained experimental data directly correspond with the pseudo-second order kinetic model. This is confirmed by the high value of correlation coefficient r2. What is more important, the amount of adsorbed dyes (qe,cal) calculated with the use of pseudo-second order kinetic model better match the obtained results (qe,exp), regardless of the type and concentration of

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Małgorzata Norman dye solution (Table 5). On the other hand, the quantity of dye adsorbed at equilibrium, computed from the pseudo-first order model deviates significantly from the experimental values.

Table 5. Parameters of kinetic models of dye adsorption on Hippospongia communis skeletons.

Kinetics model PFO PSO

Parameters qe,exp k (mg/g) q k q 2 e,cal 1 r2 e,cal (g/mg r2 Dye (mg/g) (1/min) (mg/g) min) concentration (mg/L)

25 2.49 3.80 0.044 0.970 2.810 0.029 0.979

50 4.86 4.23 0.116 0.855 4.951 0.092 0.999 carmine

75 6.84 7.23 0.100 0.986 6.869 0.382 0.999

1000 129.9 45.44 0.011 0.853 129.34 0.0013 0.997

2000 245.9 195.9 0.014 0.959 259.45 0.0002 0.992 anthocyanins 3000 341.2 183.5 0.018 0.958 347.44 0.0004 0.999

100 24.84 0.17 0.006 0.003 24.87 0.102 0.999

200 49.73 14.62 0.084 0.327 49.83 0.010 0.997 chlorphyllin 300 74.57 12.80 0.076 0.404 75.85 0.009 0.999

50 12.50 5.48 0.034 0.988 12.66 0.021 0.999

100 24.98 7.21 0.020 0.811 25.32 0.009 0.999 copper

phthalocyanine 200 48.93 30.41 0.012 0.933 50.65 0.001 0.992

The above described results of adsorption experiments and their mathematical description give information about affinity of Hippospongia communis spongin skeleton to different dyes (natural and synthetic). Desorption tests were also conducted and results

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Małgorzata Norman suggests that dyes were deposited permanently on the spongin-based sponge skeleton. According to the results it could be concluded that sponginous scaffold is a support with good adsorption properties. By adjusting process conditions, it is possible to achieve high affinity towards dyes representing different types of chemical structures (anthraquinone, flavonoids, porphyrin and phthalocyanine).

The second important aspect of the realized studies was comprehensive physicochemical and morphological characterization of both sponginous skeleton of Hippospongia communis and obtained products. The conducted elemental, spectral, thermal and structural analyses provided the information about spongin. Moreover, the obtained results indirectly confirmed the efficiency of the adsorption process and indicated the possible mechanism of interaction between adsorbent and adsorbate. The results from energy dispersive spectroscopy (EDS) and elemental analysis, presented in publications 2, 3, 4 and 5, confirmed the expected content of carbon, nitrogen, hydrogen and oxygen in spongin. Presence of sulfur indicates that amino acid - cysteine is one of the component of this material. Halogens were also detected, which verified the presence of iodotyrosine, another amino acid characteristic for spongin. The occurrence of elements at low concentration (Si, Al, Ca, Mg, Na, K) is in accordance with previously published reports [32]. X-ray photoelectron spectroscopy (XPS) was used to determine the exact chemical states of the species on the surface of the spongin. The recorded binding energies for spongin suggest the presence of C–H (~283 eV); C–C, C=C, C–OH (~285 eV); N–C=N, C–O–C (~286 eV) and C=O (N–C=O, O–C=O) (~288 eV) bonds in its structure (Fig. 9).

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Fig. 9. XPS spectra of carbon, nitrogen and oxygen in spongin.

The results obtained from other spectral technique, Fourier transformed infrared spectroscopy (FTIR) stay in agreement with XPS results. The spectrum for the adsorbent (spongin from Hippospongia communis), presented in publications 1, 2, 3, 4 and 5, displays signals indicating the presence of –NH and –OH groups (at 3300 cm-1) and stretching vibrations of C–H in –CH3 and –CH2 bonds. Moreover, occurrence of several bands: C=C, C=O, CO–NH (the characteristic band of the amide groups of protein chains), C–O–C and C–O, as well as N–C=O in spongin skeleton was confirmed. In carbon nuclear magnetic resonance (13C CP MAS NMR) spectrum of Hippospongia communis skeleton the peak observed at about 180 ppm corresponds to the carbon of a carbonyl groups C=O, and that at 150 ppm to aromatic carbons. The results indicate the presence of aliphatic carbon (saturated alkanes), carbon bonded to nitrogen (C–NR2) and to oxygen (C–OH, C–OR) with signals in the 20–80 ppm range. Thermogravimetric analysis of spongin revealed that this material is thermal stable up to 150 °C.

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The products obtained after adsorption process of dyes were subjected to comprehensive physicochemical and structural analysis using the available methods and techniques. The aim of that studies was also to unequivocally confirm the effectiveness of the adsorption process and to define the character of interactions between adsorbent and adsorbate. On the FTIR spectra of hybrid material obtained after adsorption process bands originating both from spongin matrix and dyes are noticed. The most important changes (concerning adsorption of copper and iron phthalocyanine, described in publications 4 and 5, respectively) related to the stretching asymmetric vibrations of sulfonic group (–SO3-M+) occurring in the wavenumber range 1250–1140 cm-1 in the form of a broad band observed in the spectra of Hippospongia communis and product obtained after adsorption of above-mentioned dyes (Fig. 10).

Fig. 10. Results of FTIR analysis of spongin, copper phthalocyanine (CuPC) and product obtained after adsorption process, at different wavenumber range.

Analogous observation was made in case of 13C CP MAS NMR analysis. Signals attributed to characteristic bands of spongin and selected dyes are present on the spectra of hybrid material. In publication 2 signals occurring in the range 90–175 ppm come from the

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Małgorzata Norman aglycone molecule in anthocyanin. Comparing the 13C NMR spectra of marine sponge skeleton with adsorbed, SCC (publication 3), copper phthalocyanine (publication 4) and iron phthalocyanine (publication 5), respectively, with the spectra of Hippospongia communis and mentioned dyes, it is an evident that the porphyrin and phthalocyanine macrocycle carbons appear as a series of lines in the range of 120–130 ppm in the spectra of hybrid material. The efficiency of the production of hybrid material was also confirmed by analysis of the chemical composition of the obtained product using two another techniques: XPS and EDS. Results of above-mentioned analyses demonstrate the presence of additional elements, which were not detected in pure spongin. For SCC-spongin (publication 3) and CuPC-spongin (publication 4) it was copper and for FePC-spongin - iron (publication 5). Images from optical and scanning electron microscope revealed the three- dimensional, anastomosed structure of spongin. Photos after the adsorption process illustrate the distinct dye layers on the surface of the sponge (publications 1, 2, 3, 4). Number of techniques were used to evaluate the properties of obtained dye- biopolymer material and type of interactions between the adsorbent and adsorbate. Apart from spectrophotometric calculations, other analyses indirectly confirmed the effectiveness of the adsorption process: EDS, XPS, elemental analysis, FTIR, 13C CP MAS NMR and TG/DTA. During the study course, the efficient combination of different types of dyes and spongin as well as the formation of hybrid material was confirmed. Presence of additional signals, related to the adsorbed dyes as well as sponginous scaffold of Hippospongia communis are noticed. Moreover, changes in the peaks maxima (FTIR analysis) and in values of chemical shifts (13C CP MAS NMR) of characteristic bands are observed, which may suggest the chemical interactions between constituents. A possible interaction mechanism which may explain these observations is the formation of hydrogen bonds between functional groups of the components. A schematic representation of that bonds, together with above-mentioned electrostatic interactions, are presented in Fig. 11.

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Fig. 11. Proposed mechanism of interaction between chlorophyllin and sponginous Hippospongia communis skeleton [29].

The considerable part of the research was evaluation of practical properties of obtained materials and their further application. One of the objectives of presented studies was the combination and utilization of beneficial properties of sponginous skeleton of Hippospongia communis and selected dyes. The obtained results are presented below. Because of its non-toxicity and antiradical properties naturally occurring anthocyanin has a potential for the utilization as a harmless coloring material. Its color instability under, for example, light irradiation limits its practical use. To the wider use of the anthocyanin, the immobilization on the inorganic host for an easy handling as well as the improvement of the stability is required. The impregnation of spongin skeleton with antioxidant substances could have a positive effect in both application areas, due to free radical scavenging properties, as in the case of anthocyanin dye. The adsorbed anthocyanin exhibits antioxidant properties at level compared with dye solution. This could provide a better chemical system in terms of stability without loss of bioavailability. The system obtained and described in 2 publication: natural dye - anthocyanins, adsorbed on skeleton of Hippospongia communis has antioxidant properties, as verified by the modified version of the Brand-Williams method (reduction of DPPH - 1,1-diphenyl-2-picrylhydrazyl radical to DPPH-H: 1,1-diphenyl-2-picrylhydrazine) (Fig. 12). More than 95% of radical scavenging was obtained after 30 min of the process. Different concentrations of dye solution and

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Małgorzata Norman anthocyanin-spongin with different dye content were investigated. The equivalent of Trolox was calculated to compare the results obtained with other previously described in the literature.

Fig. 12. Scheme of antioxidant activity of anthocyanin-spongin material and changes in absorbance of DPPH solutions after reaction with dye–marine sponge skeleton hybrid material (obtained from 3–10 g/L dye solution) (image copyrights – Elsevier) [28].

Numbers of bioactivities are ascribed to chlorophyllin, dye used as an adsorbate in publication 3. Knowing antibacterial properties of chlorophyllin, decision was made to test them in combination with spongin skeleton. The antibacterial activity was evaluated based on the diameter of the zone in which growth of the selected bacteria strain was inhibited. Tetracycline was used as a positive control. The results demonstrate that the marine sponge skeleton/SCC hybrid material reduced the growth of the Gram-positive microorganism Straphylococcus aureus, and the effect increased with increased concentration of SCC in hydrid material, as expected. The basic advantage of the received system in comparison to pure dye is its insolubility, which provides the possibility of reusing it. It is important in terms of potential application, among which attention should be given to preparation of wound dressing. There are literature reports concerning application of sponges (synthetic and plant ones) for wound management to promote healing of acute and chronic wounds [95–97]. The same studies pointed out the risk of Straphylococcus aureus infection [98]. The more important is that Straphylococcus aureus strain is a bacterium causing hospital- acquired infection.

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Małgorzata Norman

The essential part of the publication 4, in which the copper phthalocyanine was adsorbed onto marine sponge skeleton, was the evaluation of catalytic properties of obtained product in organic compound, synthetic dye Rhodamine B, degradation. The obtained results confirmed that the catalytic properties of CuPC are retained in combination with spongin and that Hippospongia communis skeleton is a good support for catalyst. In the presented study the combined approach for Rhodamine B degradation was involved: photochemical systems based on photosensitizers absorbing UV light (CuPC) which works as catalyst and external oxidant, H2O2, which form strong oxidizing species, like OH∙, that react directly with the molecules of Rhodamine B. The hydroxyl radicals could be produced in different ways: - during photodissociation ∙ H2O2 + hv → 2 OH - in reaction with metalphthalocyanine n n+1 − ∙ M PC + H2O2 → M PC + OH + OH - in reaction with metalphthalocyanine and light MPC + hv → FMPC → FMPC+ + e− − ∙ ∗ − e + H2O2 → OH + OH

The synergistic effect of all above-mentioned factor caused fast and almost complete (up to 95%) decomposition of Rhodamine B (Fig. 13). The mechanism of that process, based on the literature reports, was demonstrated.

Fig. 13. Rhodamine B concentration change, as a function of time, under various experimental conditions and schematic view of UV reactor.

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The promising results obtained for copper phthalocyanine incline to extend the studies and test another metalphthalocyanine in catalysis process. In publication 5 catalytic performance of obtained product, iron phthalocyanine-spongin scaffold hybrid material was evaluated. The common, aromatic water pollution phenol, as well as their halogenated derivatives chloro- and fluorophenol and bisphenol A (BPA) were taken as substances to be degraded (Fig. 14).

Fig. 14. Schematic representation of BPA, phenol, chloro- and fluorophenol degradation [33].

The effect of time, presence of hydrogen peroxide, ultraviolet irradiation, adsorption and catalyst addition on degradation efficiency was evaluated. Similar to previously described publication 4 the best results were obtained when all this factors worked together. The synergistic effect as well as degradation kinetics, according to pseudo-first and pseudo-second order model was calculated. Complete degradation of bisphenol A in concentration of 50 mg/L within 50 min took place. 2 mg/L solution of phenol, chlorophenol and fluorophenol were fully decomposed within 40 min. A significant part of the work was identification of degradation intermediates and products. Based on the products identified by high-performance liquid chromatography/mass spectrometry (HPLC-MS) and literature research the possible degradation mechanism and pathways were proposed, featuring a series of steps including cleavage of C–C bonds and oxidation. Bisphenol A undergoes a series of steps, including oxidation of the phenolic ring and oxidative scission at the sp3 carbon atom interconnecting two phenolic rings, leading to catechols and quinones. In the case of phenol and its derivatives, catechols are formed. Moreover, for chlorophenol and fluorophenol, dimer compounds are observed (Fig. 15).

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Małgorzata Norman

Fig. 15. Identified degradation product of BPA, phenol, chloro- and fluorophenol [33].

Dyeing of marine sponges was previously described by some patents [99]. However, the dyeing conditions presented there are radical and the process is multi-staged. The presented publications confirmed the promising properties of spongin skeleton as dye adsorbent, requiring no special preparation of a sponge as well as mild conditions of adsorption process. The preparation of the sponge/dye skeleton systems allowed to combine the functional properties of the selected dyes with the thermally and mechanically resistant support of natural origin and the creation of products with unique physicochemical properties and the possibility of finding interesting application. Moreover, the benefits of spongin scaffold, like three-dimensional, anastomosed architecture, their affinity to different structures of dyes and high sorption capacity make this material a promising candidate for catalyst support.

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6. Summary

The presented doctoral thesis is focused on the use of sponginous marine sponge skeleton as a support for dye adsorption. The preparation and properties of the designed dye/spongin material have been investigated in detail for the first time.

A detailed analysis of the adsorption efficiency as well as morphological and physicochemical properties of obtained materials were made. There are different groups of natural dyes: antraquinones, flavonoids or porphyrins and examples of them: carmine, anthocyanin and chlorophyllin were tested as adsorbates. Synthetic phthalocyanine-based compounds were also examined. As it was shown, spongin skeleton of Hippospongia communis exhibit affinity towards dyes of natural and synthetic origin (phthalocyanines of copper (II) and iron (III)). The high sorption capacity is an effect of structure of skeleton as well as chemical composition of spongin. Results of analysis confirmed the effectiveness of adsorption process, moreover attempts were made to determine the mechanism of interaction between dyes and sponge skeleton. Influence of several factors on adsorption efficiency was proven. Among time, initial dye concentration and temperature, the ionic strength and pH were the crucial ones, which suggest that electrostatic interaction appears between functional groups of spongin and dyes. Furthermore, this observation stays in agreement with the results of spectroscopic analyses. Based on the results of FTIR and 13C CP MAS NMR, it should be noted that the small shifts in the maximum wavenumbers of certain peaks (–OH and –NH groups, C=O bonds) may indicate that these interactions occur. Formation of hydrogen bonds is also possible, because there are no significant changes in spectra before and after adsorption process. From a scientific point of view the determination of the mechanism of interaction between the adsorbent and the adsorbate was essential. Several analyses of physicochemical and morphological studies were carried out for this purpose. In addition, the analysis of the results of these methods provided information on the spongin itself, never before presented in the literature. The adsorption of selected dyes on the surface of marine sponge skeleton may be conducted using relatively simple method with high efficiency and this process may be controlled by adjusting the basic parameter. Experimental data of adsorption process correspond to a pseudo-second order kinetic model for all dyes tested. In case of

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Małgorzata Norman adsorption isotherms, there is no unambiguous indication for Langmuir or Freundlich model, which may suggest a complex adsorption mechanism. It was proven that properties of functionalized spongin skeleton affect the application of these material. Spongin skeleton preserves the important properties of immobilized dyes and provides chemical and mechanical improvements to the produced material. Hence, the efficient approach for obtaining a 3D hybrid material of designed properties was proposed. Novel, functional dye/biopolymer hybrid materials exhibit antioxidant and antimicrobial properties against bacteria strain Straphyllococus aureus were produced, like heterogenous catalyst, spongin with adsorbed metal phthalocyanines. The catalytic properties of these material were examined in Rhodamine B as well as other harmful model organic waste (phenol, chloro- and fluorophenol, bisphenol A) degradation. The synergistic effect of adsorption, catalysis as well as ultraviolet irradiation and oxidation was confirmed. The listed achievements confirm the practical character of the realized studies and introduce significant novelty to the field associated production, characterization and practical application of materials based on marine sponge skeletons. The presented studies contribute to the knowledge about marine sponges and are located in the context of the contemporary research about biomaterials. The simple procedure of creation of new dye-spongin product increases the possibilities of utilizing this material and points out perspectives for their further development and practical use.

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[99] M. Cohn, Method of dyeing sponges, U.S. 2056166 A patent, 1934.

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Materials 2015, 8, 96-116; doi:10.3390/ma8010096 OPEN ACCESS materials ISSN 1996-1944 www.mdpi.com/journal/materials Article Adsorption of C.I. Natural Red 4 onto Spongin Skeleton of Marine Demosponge

Małgorzata Norman 1, Przemysław Bartczak 1, Jakub Zdarta 1, Włodzimierz Tylus 2, Tomasz Szatkowski 1, Allison L. Stelling 3, Hermann Ehrlich 4 and Teofil Jesionowski 1,*

1 Institute of Chemical Technology and Engineering, Faculty of Chemical Technology, Poznan University of Technology, Berdychowo 4, Poznan 60965, Poland; E-Mails: [email protected] (M.N.); [email protected] (P.B.); [email protected] (J.Z.); [email protected] (T.S.) 2 Institute of Inorganic Technology and Mineral Fertilizers, Technical University of Wroclaw, Smoluchowskiego 25, Wroclaw 50372, Poland; E-Mail: [email protected] 3 Department of Mechanical Engineering and Materials Science, Center for Materials Genomics, Duke University, 144 Hudson Hall, Durham, NC 27708, USA; E-Mail: [email protected] 4 Institute of Experimental Physics, Technische Universität Bergakademie Freiberg, Leipziger 23, Freiberg 09599, Germany; E-Mail: [email protected]

* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +48-61-665-3720; Fax: +48-61-665-3649.

Academic Editor: Harold Freeman

Received: 31 August 2014 / Accepted: 18 December 2014 / Published: 29 December 2014

Abstract: C.I. Natural Red 4 dye, also known as carmine or cochineal, was adsorbed onto the surface of spongin-based fibrous skeleton of Hippospongia communis marine demosponge for the first time. The influence of the initial concentration of dye, the contact time, and the pH of the solution on the adsorption process was investigated. The results presented here confirm the effectiveness of the proposed method for developing a novel dye/biopolymer hybrid material. The kinetics of the adsorption of carmine onto a marine sponge were also determined. The experimental data correspond directly to a pseudo-second-order model for adsorption kinetics (r2 = 0.979–0.999). The hybrid product was subjected to various types of analysis (FT-IR, Raman, 13C CP/MAS NMR, XPS) to investigate the nature of the interactions between the spongin (adsorbent) and the dye (the adsorbate). The dominant interactions between the dye and spongin were found to be hydrogen bonds and Materials 2015, 8 97

electrostatic effects. Combining the dye with a spongin support resulted with a novel hybrid material that is potentially attractive for bioactive applications and drug delivery systems.

Keywords: C.I. Natural Red 4; carmine; dye adsorption; kinetic model; marine sponge; spongin; Hippospongia communis

1. Introduction

The synthetic dyes used in foodstuffs have relatively low production costs, high stability, and resistance to environmental conditions. This group of substances can be used to create a wide range of colors, as well as offering water solubility. They are also resistant to sudden changes in pH, temperature and light. In some cases, however, they may have a harmful effect on living organisms, and may contain undesirable additional substances [1,2]. Natural dyes used in foodstuffs, in turn, do not pose any risk to health, although they have weaker coloring properties and lower color intensity. They may also be sensitive to a number of factors: high temperature, changes in pH, and oxidants. Natural dyes are usually obtained by a process of extraction, purification, and concentration from plant or animal sources [3,4]. Carmine (7-α-D-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-anthracene carboxylic acid), molecular weight 492 (g/mol), also called C.I. Natural Red 4 or cochineal, is a dark red dye obtained from dried and crushed insects from the Coccidae family (scientific name: Dactylopius coccus) [5,6]. Industrial carmine is obtained by mixing carminic acid with metal salts [7]. The structure of carminic acid is based on anthraquinone with multiple hydroxyl groups, a carboxyl group, and a glucose sugar unit side chain. The molecular structure of the dye is shown in Figure 1. This dye is susceptible to thermal decomposition and photodegradation, but exhibits relatively high chemical and biological stability [8].

Figure 1. Structure of carminic acid.

As carmine is a harmless substance, it is used chiefly in the food, pharmaceuticals and cosmetics industries. It is also used as an indicator in analytical chemistry, and to a lesser extent in textiles and plastics [7]. Further uses include as a dye in microbiology, and for the modification of ion exchangers [9]. Due to the presence of its OH groups, it can form complexes with metal ions like U(VI), Th(IV), Mo(VI)) [10]. Carmine has also many biological applications: it can prevent coronary artery disease [11], it plays a role in treating Alzheimer’s disease [12], it exhibits cancer chemopreventive activity, [13] and is used in drug delivery products [14]. There exist a few publications concerning the adsorption of carmine

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or carminic acid, and they relate exclusively to inorganic adsorbents: TiO2 [15], glass beads [16], Amberlite XAD-16 resin [9], Ag nanoparticles [17], methacrylic acid (MAA) and 4-vinylpyridine [18], and multiwalled carbon nanotubes [19]. Carmine or carminic acid have also been combined with SiO2 sol-gel glass [8], hydrotalcite [20], and amorphous SiO2 [21]. Marine demosponges are representatives of the class Demospongiae that belong to the phylum Porifera [22]. The species Hippospongia communis also known as bath sponge, belongs to the Dictyoceratida order. Species of this order possess three-dimensional non-mineralized fibrous skeletons, which are composed mainly of the protein-like substance spongin. Spongin of Demosponges is very similar to collagen type XIII found in vertebrates which was confirmed by characteristic aminoacid composition [23,24]. Genomic and complementary DNA studies showed that spongin (similarly to collagen) contain the classic collagenous Gly-Xaa-Yaa motif where Hydroxyproline (Hyp) occupies any of the positions in the triplet motif, other than Gly (Glycine) position [25,26]. This biopolymer of still unknown chemical structure seems to be a naturally occurring hybrid between collagen and keratin-like proteins that contains sulfur, bromine and iodine [27–31]. Because of its unique physico-chemical, structural, and mechanical properties [32] spongin-based skeletons of bath sponges has been broadly used since ancient times in household use and medicine [33]. Nowadays, their biocompatibility [34] and specific arrangement of structural elements like pores, struts and channels offers model scaffolds for tissue engineering [35,36]. Spongin-containing marine sponges, including Mediterranean H. communis, are examples of renewable resources due to their ability to be cultivated under marine ranching conditions [37,38]. This property enhances the biomimetic potential of bath sponges as organisms, and that of spongin as a specific biological material. In contrast to the attempts to dye bath sponges with synthetic dyes, their ability to adsorb natural dyes is still not studied. Thus, Cohn in his patent [39] reported as follows: “It has been suggested, as disclosed in the English Patent to Asher 14,866 of 19 July 1905, that sponges of some unidentified type could be dyed when treated first with a metallic mordant at some unidentified temperature and then dyed in an alizarine bath at temperatures of 70–80 ° C (158–176 °F). The primary objection to sponges dyed in accordance with the suggestions in the Asher patent is that the colors are not fast, and the resulting so-called sponge bleeds when wetted with warm water. Further, mordants of the type in general use in 1905, such as the basic aluminum sulfates suggested, in order to be effective must necessarily be heated at temperatures approaching the boiling point (even if the boiling point is not actually reached). Sponges dyed by the method disclosed in this English patent do shrivel up despite the claim in the patent that shriveling is avoided when the alizarine lakes are maintained at temperatures not exceeding the 70–80 °C. However, a more serious objection than simply that the sponge becomes shriveled and cannot retain the color of the lake in which it was dyed, is that it is otherwise deleteriously affected in its physical characteristics—for instance, it loses to a large extent its elasticity, or spring, or “life”. Any temperature as high as 70–80 °C appears to destroy or at least partially close the inhalant pores, the canals, the apoyles, and the oscules, and thus interferes with the water flow through the sponge’s passageways. It has been found that bleached sponges dye much more readily than unbleached ones. Bleached sponges also require about one-half as much color as unbleached sponges do. The bleaching of the sponge apparently doubles its color absorbing qualities. Bleaching, particularly with the permanganate method proposed, gives the sponge aseptic properties and the developers used in Step I act additionally as preservatives to prevent bacteria and mold growth [39].

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To our best knowledge, there are no reports to date concerning the adsorption of natural dyes using sponginous skeletons of marine sponges as a support. The aim of the present study is to obtain interesting dye/biopolymer hybrid materials. Combining the dye with a support improves its bioavailability and its resistance to chemical and thermal degradation. This enhanced stability creates opens the door for future applications, which may include creating a biocompatible material used in drug delivery.

2. Results and Discussion

2.1. Spectrophotometric Investigation

Absorption spectra (400–900 nm) were obtained for carmine in water at different pH values. Over the analyzed pH range (3–11) the absorption maxima varied only slightly. The color of the C.I. Natural Red 4 water solution showed marginal variation, as it exists in several forms [17]. Measurements of the absorbance of C.I. Natural Red 4in a solution with pH = 7 over the full visible light range showed a maximum absorbance at 513 nm. The absorption spectrum of carmine in water undergoes a red shift (shift to a longer wavelength absorption) upon addition of metal salts [40]. Based on the literature data, for pure carminic acid this value equals 493 nm.

2.2. Effect of Contact Time and Dye Concentration

Figure 2 shows the effect of carmine concentration on the amount of the dye adsorbed (qt) on the H. communis spongin scaffold, plotted against time.

Figure 2. Adsorption capacity for C.I. Natural Red 4 onto H. communis sponge skeleton, as a function of time (results obtained in pH = 7).

The highest value of qt was obtained after reacting for 90 min. The adsorption capacities for 25, 50 and 75 mg/L of C.I. Natural Red 4 onto the marine sponge skeleton were 2.45, 4.79 and 6.84, respectively. The efficiency of C.I. Natural Red 4 adsorption decreased as the initial concentration increased, even though the quantity of dye adsorbed per unit mass of adsorbate increased. This is linked to the quantity

Materials 2015, 8 100 of molecules of the dye adsorbed on the surface of the support: the smaller the dye concentration, the more molecules present in the solution can become bound to the adsorbent. An increase in concentration leads to saturation of the active sites of the support, which reduces the efficiency of the process; because in effect a significant number of dye molecules are not adsorbed [41]. Adsorption is reduced due to the lack of sufficient available open sites to adsorb high initial concentrations of the dye [11–16]. This situation is caused by the mass transfer driving force, which increases when the initial concentration is increased, resulting in higher adsorption of dyes [42,43]. Various studies have confirmed that adsorption capacity increases as the dye concentration increases [44,45]. As can be seen from Figure 2, the quantity of dye adsorbed rose very rapidly in the course of the first few minutes of the process, for all tested concentrations of dye in solution. Similar effects were observed when carmine was absorbed onto glass beads [16]. The contact time needed for C.I. Natural Red 4 (in every initial concentration) to reach equilibrium was around 30 min. The quantity of carmine adsorbed on marine sponges increases with time, and reaches a constant value beyond which no more dye is removed from the solution. At this point, the quantity of dye desorbing from the marine sponge skeleton is in a state of dynamic equilibrium with the quantity being adsorbed onto it. This observation can be explained by the theory that diffusion onto the external surface of the adsorbent was followed by diffusion into the intra-particle matrix to attain equilibrium [46].

2.3. Effect of pH

The manner in which the pH of the environment affects the efficiency of adsorption of the dye from solution was also examined. The process was carried out at pH = 3, 5, 7 and 9, for initial dye concentrations of 25, 50 and 75 mg/L. Adsorption tests were performed over 30 min. It was observed that an increase in the acidity of the solution increases the efficiency of the adsorption of C.I. Natural Red 4. The quantity of dye adsorbed from a 25 mg/L solution increases from 1.42 mg/g (56.7%) at pH = 7 to 2.45 mg/g (98.1%) at pH = 3. In a basic solution, however, the efficiency of the adsorption process is zero. The same patterns were observed for the other carmine concentrations. + This is linked to the protonation of the neutral –NH2 amine groups in the protein scaffold to form –NH3 cationic groups. Under these conditions the process of adsorption of the dye occurs via electrostatic + interactions. An increase in the pH leads to deprotonation of –NH3 groups, and in effect only hydrogen bonds [47] are formed between the support and the dye, reducing the efficiency of adsorption. Similar adsorption behavior as the pH is varied has been reported in the literature for compounds containing NH2 groups [42,44,48]. The results are presented in Table 1.

Table 1. Effect of pH on adsorption capacity for C.I. Natural Red 4 onto marine sponge. Dye concentration (mg/L) pH 25 50 75

Experimental qt (mg/g) 3 2.45 5.00 7.50 5 1.50 3.62 6.71 7 1.42 3.42 5.99 9 0.00 0.00 0.00

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A similar range of pH values was used during carminic acid impregnation of the resin Amberlite XAD-16. A continuous decrease in impregnation efficiency at both pH > 3 and pH < 3 was observed. The lowest impregnation efficiency of 89% was at pH = 8 [9]. However, the required time needed for completing the impregnating process was found to be at least 3 h. A similar effect of pH on carminic acid adsorption is also described in [17].

2.4. Desorption Test

Desorption tests for samples containing C.I. Natural Red 4 were carried out at different pH values (7 and 9). In contrast with the adsorption tests performed at varying pH, the results in this case were not affected by the acidity of the environment. The efficiency of the process was 13.4% at pH = 7, and 12.8% at pH = 9. It was observed, however, that the desorption percentage was greater when the dye was washed from samples that contained a greater quantity of dye following the adsorption process. In the case of marine sponge skeleton pieces containing 4.54 mg/g of C.I. Natural Red 4 the desorption percentage was 18.8%, while from samples containing 2.27 mg/g the percentage was 4.7%.

2.5. Kinetic Analysis

To investigate the kinetics of the adsorption process, pseudo-first-order (PFO) and pseudo-second-order (PSO) models were used. These investigations make it possible to describe the controlling mechanism of the adsorption process. A pseudo-first-order equation is:

(1) 푘 where q and q (mg/g) are the quantitieslog푞e − of 푞푡 dye= logadsorbed푞e − at time· t 푡 (min) and at equilibrium, and k t e . 1 (1/min) is the rate constant of pseudo-first-order sorption. The pseudo-first-order model refers to an adsorption process in which sorption proceeds by diffusion through a boundary. A pseudo-second-order equation is:

(2) 푡 = + · 푡 where k2 (g/mg·min) is the pseudo-second-order푞푡 푘 rate푞e constant.푞e When the adsorption process proceeds according to a pseudo-second-order model, the limiting step may be chemical adsorption involving valent forces through the sharing or exchange of electrons between the sorbent and adsorbate [49]. In [16], the adsorption kinetics of C.I. Natural Red 4 were described using the kinetic approximations proposed by McKay and Boyd. While adsorption is possible as a result of interaction of the functional groups of carmine and glass beads, its rate is controlled by film and particle diffusion. In this study, the kinetics of the adsorption process were described using different pseudo-first-order and pseudo-second-order models. The equilibrium adsorption capacity (qe) and adsorption rate constant

(k1) (Table 2) were computed experimentally from a plot of log(qe – qt) against t (Figure 3). The coefficient of correlation (r2) obtained when a pseudo-first-order kinetic model is used to describe the adsorption of C.I. Natural Red 4 (in concentrations of 25–75 mg/L) lies in the range 0.855–0.986.

The values of adsorption capacity (qe,cal) computed from the pseudo-first-order kinetic model deviated

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significantly from the experimental capacities (qe,exp). This indicates that the pseudo-first-order kinetic model does not fit well to the experimental data. A significantly better model describing the kinetics of adsorption of C.I. Natural Red 4 onto the marine sponge is the pseudo-second-order kinetic model

(Figure 4). The pseudo-second-order model k1 value is lower than k2, indicating that the pseudo-second-order equation better describes the adsorption process.

Table 2. Pseudo-first-order and pseudo-second-order kinetic parameters and coefficient of determination for adsorption of C.I. Natural Red 4 onto marine sponge. Parameters Concentration of dye (mg/L) Type of kinetics Symbol Units 25 50 75

qe,exp mg/g 2.492 4.858 6.836

qe,cal mg/g 3.799 4.229 7.232

Pseudo-first-order k1 1/min 0.044 0.116 0.100 r2 – 0.970 0.855 0.986

qe.cal mg/g 2.810 4.951 6.869 k 1/min 0.029 0.092 0.382 Pseudo-second-order 2 r2 – 0.979 0.999 0.999 h mg/g min 0.232 2.246 18.023

Figure 3. Pseudo-first-order kinetic fit for adsorption of C.I. Natural Red 4 onto H. communis sponge skeleton.

Figure 4. Pseudo-second-order kinetic fit for adsorption of C.I. Natural Red 4 onto H. communis sponge skeleton.

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2.6. Adsorption Isotherms

Adsorption isotherm is a graphical representation indicating the relation between the mass of the adsorbed dye per mass of the used adsorbent, and liquid phase of dye at equilibrium concentration. Based on the experimental data adsorption isotherms were determined based using the Freundlich [42] and Langmuir [41] models.

The plot of (qe) versus (Ce) for the adsorption isotherms of C.I. Natural Red 4 onto the Demosponge skeleton is presented in Figure 5. Table 3 shows the parameters for the Freundlich and Langmuir isotherms. The Freundlich equation is given as:

(3) 푛 where Ce is the equilibrium concentration of the푞e =dye 퐾 F(mg/L),· 퐶e qe is the quantity of the adsorbed dye per mass of adsorbent (mg/g), and KF (mg/g) and n are the Freundlich constants. The KF and n values can be estimated from the intercept and slope of a linear plot of logqe versus logCe.

Figure 5. Fitting of the Langmuir and Freundlich isotherm models to equilibrium results of C.I. Natural Red 4 adsorbed onto marine sponge skeleton.

Table 3. Freundlich and Langmuir isotherms constants for C.I. Natural Red 4 adsorbed onto marine sponge skeleton. Langmuir parameters Freundlich parameters 2 2 R qm (mg/g) b (L/mg) R KF (mg/g) n 0.995 18.55 0.034 0.877 3.601 4.195

The value n determines the degree of nonlinearity between the solution concentration and adsorption: a value n < 1 indicates a normal isotherm, while n > 1 indicates a cooperative adsorption. The value n computed from Freundlich’s equation for the adsorption of C.I. Natural Red 4 onto the marine sponge skeleton is equal to 4.195. The equation of a non-linear Langmuir isotherm model takes the following form:

(4) 푞m · 푏 · 퐶e 푞e = + 푏 · 퐶e Materials 2015, 8 104

where Ce is the equilibrium concentration in the solution (mg/L), qm is the maximum adsorption capacity and b is the Langmuir constant (L/mg), calculated from the intercepts and slopes of linear plots of

Ce/qe versus Ce. The sorption capacity calculated using the Langmuir model was equal to 18.55 mg/g. Comparing the isotherms’ parameters it can be concluded that the experimental data definitely resemble the Langmuir model, which is borne out by the high correlation coefficient (R2 = 0.995).

2.7. FT-IR

To confirm the effectiveness of adsorption of C.I. Natural Red 4 onto spongin fibers, FT-IR spectra of the products were taken to check for the presence of characteristic functional groups. Detailed investigations were performed for the H. communis sponge skeleton and C.I. Natural Red 4. Additional measurements were made for the dye/spongin hybrid material obtained from an initial dye concentration of 50 mg/L, and a reaction time of 30 min. Details of the bands present in the spectra, with their wavenumbers and band assignments, are given in Table 4.

Table 4. FT-IR characteristic wavelengths for C.I. C.I. Natural Red 4, marine sponge and hybrid material (dye solution 50 mg/L, contact time 30 min, pH = 7). C.I. Natural Hippospongia Dye/Biopolymer Vibrational Red 4 communis skeleton hybrid material assignment 3400 3410 3415 –OH stretching – 3300 3310 –NH stretching 2930 2930 2930 –CH2, –CH3 stretching 1650 1630 1655 C=O stretching

1560 – 1560 C=CAr stretching – 1520 1525 –NH deformational – 1460 1460 –CH scissors 1400 1400 1405 –OH stretching – 1250 1250 C–N stretching 1080 1080 1075 C–O–C stretching 1020 1020 1020 C–O stretching 900 – 907 –OH bending 660 – 660 –CHAr deformational 520 – 525

The spectrum for the adsorbent (Hippospongia communis) displays signals indicating the presence of –NH bonds (at 3300 cm−1 and 1520 cm−1) and C–N bonds (at wavenumber 1520 cm−1), which are part of the proteinaceous (spongin) skeleton of the sponges. The vibrations generating these bands occur only in the structure of the spongin; they are not observed in the dye molecule. −1 −1 −1 The bands at 1560 cm (C=CAr stretching vibrations), 660 cm and 520 cm (–CHAr deformational vibrations) are found as original signals, and are only observed in C.I. Natural Red 4. There is also a clear signal at wavenumber 900 cm−1 due to bending vibrations of OH groups from carboxyl group. All of these data are in agreement with the literature [6,15,19].

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The spectrum of the dye/biopolymer hybrid material reveals the presence of signals characteristic for both the adsorbent and carmine. However, the maxima are shifted in the direction of higher wavenumber values for the stretching vibrations of hydroxyl groups (3415 cm−1 and 1405 cm−1) and the stretching vibrations of C=O groups (1655 cm−1). The same occurs for the bands generated by stretching and deformational vibrations of –NH groups in the sponges (at 3310 cm−1 and 1525 cm−1) and the bending vibrations of carboxylic–OH groups in the dye (at 907 cm−1). Apart from the shift in the maxima, certain signals are found to be more intense in the spectrum of the hybrid material. This is particularly visible in the case of the bands assigned to hydroxyl groups (3415 cm−1 and 1405 cm−1), stretching vibrations −1 −1 from –CH2 and –CH3 groups (2930 cm ), and stretching vibrations due to C=O (1630 cm ). This is due to the fact that these functional groups are present in both starting materials. A further factor may be the mechanism of adsorption of the dye on the sponge surface (the formation of hydrogen bonds between their surface groups, which are responsible for the observed chemical shifts) [50]. The results are presented in Figure 6.

Figure 6. FT-IR spectra of C.I. Natural Red 4, marine sponge and hybrid material (dye solution 50 mg/L, contact time 30 min, pH = 7).

2.8. XPS

The chemical structures of carmine, marine sponge skeleton and the dye/biopolymer hybrid were also studied by XPS spectroscopy. This was used to determine the relative quantities of dye adsorbed on the spongin biopolymer surface. Figure 7 shows XPS spectra of the dye, the spongin, and a selected dye/biopolymer material. Table 5 contains the results of quantitative analysis of the samples. Due to the high similarity of the high-resolution spectra for the main components C 1s, O 1s and N 1s in the support and the dye, they could not be used as a basis for determining the adsorbed quantity of dye. In addition, no changes were found in the bond energies of the aforementioned components following impregnation of the support with dye. Among the elements potassium, zinc, silicon and sulphur identified in the dye (Table 5), only zinc was detected on the surface of the dye/biopolymer hybrid. The quantity of this element rose from 0.16% to 0.31% in samples 1–3 (Table 6), in accordance with the sequence of increasing concentration of dye in the initial solution. The quantity of zinc on the surface of the samples was determined using the main Auger Zn

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LMM line of photoelectrons (Figure 8), in view of their superior quality compared with the line Zn 2p3/2 (at such low surface concentrations of zinc). The value of the modified Auger α’ parameter for Zn LMM and Zn 2p3/2 electrons for the pure dye and the tested samples was approximately 2010 eV, indicating the presence of ZnO.

Figure 7. XPS spectra of: (a) C.I. Natural Red 4; (b) H. communis sponge skeleton; (c) hybrid material (obtained from 75 mg/L dye solution, contact time 30 min, pH = 3).

Table 5. Surface composition and relative concentration of elements obtained by XPS analysis of the marine sponge skeleton, C.I. Natural Red 4, and selected hybrid materials. Sample C N O K S Zn Si Hippospongia communis 72.54 5.25 22.21 – – – – C.I. Natural Red 4 57.90 4.17 31.43 3.60 0.83 2.05 – Sample 1 (25 mg/L, 30 min, pH = 3) 66.63 7.37 24.44 – – 0.16 1.40 Sample 2 (50 mg/L, 30 min, pH = 3) 67.59 6.08 24.75 – – 0.18 1.40 Sample 3 (75 mg/L, 30 min, pH = 3) 68.52 4.85 26.32 – – 0.31 –

Table 6. Zinc content in selected dye/biopolymer samples (dye solution 1:25 mg/L; 2:50 mg/L; 3:75 mg/L; contact time 30 min, pH = 3). Sample 1 2 3 Zn, % at. 0.16 0.18 0.31 Zn:N 0.022 0.030 0.064

Clearer confirmation of the correlation between the concentration of dye in the initial solution and its content in samples 1–3 is provided by analysis of the Zn:N ratio, which increased from 0.022 in sample 1 to 0.062 in sample 3. The relatively greater increase found for the Zn:N ratio than for the absolute content of Zn results from higher coverage of the surface by the dye. It should be noted that the greater part of the nitrogen recorded in samples 1–3 came from the biopolymer (>90%, estimated from the decrease in Zn content in the samples compared with the pure dye); also the N 1s bond energies for samples 1–3 and the biopolymer were identical at 399.75 eV, compared with 399.30 eV for C.I. Natural Red 4.

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Figure 8. High-resolution XPS spectrum of Zn LMM photoelectrons: (a) dye; (b) sample 1 (dye solution 25 mg/L, contact time 30 min, pH = 3); (c) sample 3 (dye solution 75 mg/L, contact time 30 min, pH = 3).

2.9. NMR

The effectiveness of the modification was also verified by means of 13C CP/MAS NMR. The results proved that the carmine/spongin interactions are of a chemical nature. Figure 8 shows a 13C CP/MAS NMR spectrum of C.I. Natural Red 4, spongin and the obtained hybrid material. Attribution of peaks for the dye was made according to [21,51]. The most intense signals, occurring in the range 60–80 ppm, come from the glucose residue, and the signal at 20.4 ppm is from a CH3 group. The chemical shifts observed above 100 ppm are attributed to aromatic carbons (C=C bonds), and that at δ = 178.2 ppm to the carbon of a carboxyl group. The 13C CP/MAS NMR spectrum of H. communis spongin indicates the presence of aliphatic carbon (saturated alkanes), as well as carbon bonded to nitrogen (C–NR2) and to oxygen (C–OH and C–OR) with signals in the 20–80 ppm range. There is also a marked signal at δ = 174.3, which is characteristic of carbon occurring in a carboxyl group or its derivatives [30,52]. Marine sponge spongin has an inexact chemical structure, where each resonance represents not just one but a range of chemical environments. Due to the lack of NMR data, the attachment position was not elucidated but only proposed. However, comparing the spectrum obtained for the spongin with that of collagen [53], many similarities are observed, indicating the high degree of similarity of their structures. The spectrum for one of the dye/biopolymer materials is shown in Figure 9. It contains a number of signals which are not seen in the spectrum of the adsorbent: δ = 170.9, 69.8, 49.8, 38.5, 21.5 ppm. In the range 100–150 ppm, as the quantity of adsorbed dye increases, peaks corresponding to aromatic carbon become visible. Moreover, comparing the spectra of the hybrid product and marine sponge skeleton (taken as a reference sample), changes in the intensities, positions and widths of other signals are observed. Unfortunately, some resonances are difficult to observe because of the low signal/noise ratio.

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Figure 9. 13C CP/MAS NMR spectra of: (a) C.I. Natural Red 4; (b) hybrid material (dye solution 75 mg/L; contact time 30 min, pH = 3); (c) H. communis spongin.

2.10. Raman Spectroscopy

The Raman spectra of carmine, marine sponge skeleton and dye/biopolymer material are shown in Figure 10. In the Raman spectroscopy results, as in the case of NMR spectra, the spectrum for the sponge material (spongin) is similar to that of collagen [54]. The bands at 2883 cm−1 and 2938 cm−1 can be attributed to the weak stretching mode of OH and −1 medium-strong asymmetric stretching of CH3. The signal at 1671 cm corresponds to the weak-medium stretching mode of C=O, that at 1448 cm−1 to weak N–H bending, and that at 1281 cm−1 to weak-medium stretching of C–N. The signals between 1100 cm−1 and 1000 cm−1 can be attributed to weak asymmetric stretching of C–O–C. Some additional signals, originating from CH and CH3 in the glucose residue of carmine, are observed in two spectral regions (1350–1050 cm−1 and 880–680 cm−1) [17,55]. The increase in the intensity of signals at 1670 cm−1and 1452 cm−1, and the appearance of peaks at around 550 cm−1 (skeletal vibration), are associated with stretching vibrations in benzene rings in the dye. In the case of the dye/biopolymer hybrid material, the results of Raman spectra analysis were similar to those for the marine sponge. Analysis of the spectra did not reveal any new bands; however, the bands’ intensity changed as a consequence of overlapping of the bands characteristic of marine sponge and dye. We conclude that the dye interacts by hydrogen bonding with the hydroxyl and carbonyl groups of spongin.

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Figure 10. Raman spectra of H. communis spongin and selected hybrid materials (dye solution 100 mg/L, pH = 7, contact time 15 and 60 min).

2.11. Thermal Stability

Thermal analysis is the main method for determining the thermal properties of chemical substances. The results of these measurements, which give information about the thermal stability of a substance, are among the most important parameters determining the range of potential applications for materials. The thermal decomposition profiles of marine sponge skeleton and C.I. Natural Red 4 are shown in Figure 11. As mentioned above, both sponge skeletons consist of the protein-like spongin. It is observed from the TG curve that the thermal degradation of H. communis spongin takes place in two stages. The first stage, in the range 80–110 °C, is associated with the evaporation of water. The second stage, involving considerable mass loss (60%–70%), is observed in a temperature range from 210 °C to 410 ° C ; and can be associated primarily with the thermal decomposition of the organic phase [56]. In the range 600–1000 °C there is another small drop in mass (from 77% to 82%), which may be associated with combustion of the organic matrix [57].

Figure 11. Thermal analysis of C.I. Natural Red 4, H. communis spongin and selected hybrid materials (dye solution 25 mg/L, pH = 7, reaction time 1, 15 and 30 min).

There are no thermogravimetric analysis data available for carmine, but it is known that the C.I. Natural Red 4 molecule consists of anthraquinone and glucose. The first mass loss (10%) is caused

Materials 2015, 8 110 by loss of water. According to [58], the first obvious peak in the TG curve for glucose pyrolysis occurred at a temperature of 239 °C. The maximum mass loss rate of glucose occurred at 301 °C. Further observed mass loss may be caused by thermal degradation of the aromatic part of the carmine structure [59]. Thermal stability measurements were also performed for selected dye/biopolymer hybrid materials. The mass loss profiles of these samples are similar to those obtained for the marine sponge spongin. The thermogravimetric curves, irrespective of the quantity of adsorbed dye, show mass loss caused by the transformations that occur as the temperature increases. However, as the amount of dye in the hybrid material increases, its thermal stability increases. The mass loss of the initial sample at 800 °C, for a product obtained after 1 min of adsorption contact time, was 47%; while for 15 min it was 42%, and for 60 min equals 24%. The increase in the stability of the hybrid material compared with the native adsorbent and with C.I. Natural Red 4 can be attributed to hydrogen bonds and electrostatic interactions formed between hydroxyl groups of the marine sponge spongin and the dye. The observed temperature peaks of mass loss in the TG curves for H. communis spongin, C.I. Natural Red 4 and the dye/adsorbent hybrid material made it possible to verify the difference between these compounds. This serves to confirm the effectiveness of the method for obtaining the new composite material.

2.12. SEM

The SEM images in Figure 12 show the H. communis skeletal fibers and a selected dye/biopolymer hybrid material. Analysis of SEM images taken before and after adsorption confirmed that the deposition of C.I. Natural Red 4 onto the marine sponge skeletal fibers had taken place. The SEM images reveal the presence of dye microparticles (Figure 12b,c).

Figure 12. Scanning electron microscopy (SEM) micrographs of: (a) H. communis fiber (after demineralization); and (b,c) a selected dye/biopolymer hybrid material (50 mg/L, contact time 30 min, pH = 7) at different magnifications.

3. Experimental Section

3.1. Materials

Dried marine sponges of the species Hippospongia communis (Demospongiae) collected in Tunesian coastal waters were purchased from INTIB GmbH (Freiberg, Germany). The preparation of the adsorbent involved washing the dry sponge with fresh water to remove salts, and immersing it completely in 3 M HCl solution for 72 h at room temperature to dissolve foreign calcium carbonate—containing debris. The material was then rinsed with distilled water until the pH of the washing solution reached 6.5, and finally dried for 24 h at 50 °C in a drying oven.

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C.I. Natural Red 4 in powder form was purchased from Sigma-Aldrich (St. Louis, MO, USA). The stock solution was prepared by dissolving an accurately weighed 500 mg portion of dye in 1000 mL of distilled water. Experimental solutions of desired concentration were obtained by successive dilutions with distilled water.

3.2. Adsorption and Desorption Experiments

Batch experiments (at 25 °C) were performed to investigate the effect of contact time, and to determine the kinetic parameters. Adsorption experiments were performed using 250 mL glass bottles containing 0.5 g of marine sponge skeleton as prepared above and 50 mL of the dye solution. The initial concentrations of the dye were 25, 50 and 75 mg/L, respectively. After different time intervals the samples were filtered off under vacuum and taken for spectrophotometric evaluation (Spekol 1200, Analytik Jena, Jena, Germany) at the maximum absorbance wavelength

513 nm. Dye concentration in the adsorbent phase at a specific time (qt), and the adsorption efficiency (E%), were calculated as:

(5) 퐶 − 퐶푡 · 푉 푞푡 = 푚 (6) 퐶 − 퐶푡 퐸% = · % where C0 and Ct are the concentrations of the dye in퐶 the solution before and after sorption respectively (mg/L), V is the volume of solution (L), and m is the mass of the support (g). The effect of pH on the adsorption of carmine from aqueous solution onto the marine sponge skeleton was investigated in a similar manner. The pH was adjusted to 3, 5, 7 and 9 using either 1 M HCl or 1 M NaOH. A desorption experiment was performed by placing 0.5 g of selected samples in a 250 mL conical flask with 50 mL of water and shaking at room temperature for 1 h. The desorption of C.I. Natural Red 4 from the hybrid material was measured by UV-Vis absorption, as described above. Adsorption isotherms were obtained by placing the samples of 0.5 g of marine sponge skeleton in a series of flasks containing 50 mL of dye solution at the desired initial concentrations (50–1500 mg/L) at room temperature. Dye concentration after 60 min of phase contact time was measured spectrophotometrically at the maximum absorbance wavelengths. The quantity of dye adsorbed at equilibrium (qe), was calculated from Equation (7):

(7) 퐶 − 퐶푡 · 푉 where C and C are the initial and equilibrium푞푡 concentration= of dye (mg/L), V is the volume of solution (L), 0 e 푚 and m is the mass of the support (g). The experimental data were used to determine Freundlich and Langmuir adsorption isotherms.

3.3. Testing of Physicochemical Properties

FT-IR spectral analysis was performed using a Vertex 70 (Bruker, Bremen, Germany). The samples were analyzed in the form of tablets, made by pressing a mixture of anhydrous KBr (ca. 0.1 g) and 1 mg

Materials 2015, 8 112 of the tested substance in a special steel ring, under a pressure of approximately 10 MPa. Analysis was performed over a wavenumber range of 400–4000 cm−1 (at a resolution of 0.5 cm−1, number of scans: 64). X-ray photoelectron spectra were obtained with a UHV/XPS/AES System (SPECS) with a PHOIBOS 100 analyzer (SPECS, Berlin, Germany) and Mg Kα anode (1253.6 eV). The background line was determined by Shirley’s method. The selected reference line was C 1s 284.8 eV (C–C, C–H). NMR analysis was performed using a DSX spectrometer (Bruker). A sample of about 100 mg was placed in a rotator, made of ZrO2, 4 mm in diameter, which enabled spinning of the sample. Centrifugation at the magic angle was performed at a spinning frequency of 8 kHz. 13C CP/MAS NMR (Cross Polarization Magic Angle Spinning Nuclear Magnetic Resonance) spectra were recorded at 100.63 MHz in a standard 4 mm MAS probe using a single pulse excitation with high power proton decoupling (pulse repetition 10 s, spinning speed 8 kHz). Raman scattering spectra were investigated in the spectral range 100–3800 cm−1 (number of scans: 1024). The non-polarized Raman spectra were recorded in a back scattering geometry, using the inVia Renishaw micro-Raman system. The inVia Raman spectrometer (Renishaw, Wotton-under-Edge, UK) enabled the recording of Raman spectra with a spatial resolution of about 1 μm. The spectral resolution was 2 cm−1. The excitation light used was a laser operating at 785 nm. The laser beam was tightly focused on the sample surface through a Leica 50 × LWD (long working distance) microscope lens with numerical aperture (NA) equal to 0.5, producing a laser beam with a diameter of about 2 μm. To prevent any damage to the sample, the excitation power was fixed at about 5 mW. The position of the microscope lens was piezoelectrically controlled during measurement. A thermogravimetric analyzer (TG/DTA/DSC, model Jupiter STA 449F3, Netzsch, (Selb, Germany) was used to investigate the effect of heat on the samples. Measurements were carried out under a flowing nitrogen atmosphere (10 cm3/min) at a heating rate of 10 °C/min over the temperature range 25–1000 °C, with an initial sample weight of approximately 5 mg. The morphology and microstructure of the samples were studied using SEM images recorded from an EVO40 scanning electron microscope (Zeiss, Oberkochen, Germany). Before testing, the samples were coated with Au for a period of 5 sec using a Balzers PV205P coater (Oerlikon Balzers Coating AG, Balzers, Liechtenstein).

4. Conclusions

Marine spongin-based demosponges have unique physicochemical properties, and as such may have many practical applications. The process of adsorption of C.I. Natural Red 4 onto H. communis was found to depend on pH and time. When the initial dye concentration increases, the adsorption capacity at equilibrium increases, while the adsorption efficiency decreases. This indicates that initial dye concentration plays an important role in the adsorption of dyes. The experimental data correspond to a pseudo-second-order kinetic model of adsorption, which indicates that the rate-controlling stage of the process involves chemical adsorption. For the measured spectra (FT-IR, Raman, 13C CP/MAS NMR, XPS) of the carmine/spongin hybrid material, only slight changes are observed relative to the spectra of the adsorbent. The lack of any significant changes suggests that there are no strong interactions between carmine and spongin. A possible interaction mechanism which may explain these observations is the formation of hydrogen

Materials 2015, 8 113 bonds between the –OH and –COOH of the dye and the marine sponge skeleton. Moreover, the results obtained for adsorption at different pH values suggest the existence of additional interactions. The highest adsorption efficiency is observed at low pH values. In future work, further studies of adsorption will be undertaken to investigate other parameters that may affect this process. These will include temperature, quantity of biosorbent and ionic strength, as well as additional analyses; and will be carried out to confirm our assumptions. The use of this novel C.I. Natural Red 4 dyed spongin skeleton for medical applications, including drug delivery, will also be studied in future experiments.

Acknowledgments

This work was financially supported by Poznan University of Technology research Grant No. 03/32/443/2014-DS-PB and DFG Grant EH 394/1-1.

Author Contributions

Małgorzata Norman was responsible for preparing and analyzing dye/biopolymer hybrid material, and obtaining results; Przemysław Bartczak did kinetic analysis; Jakub Zdarta contributed to FT-IR analysis; Włodzimierz Tylus did XPS analysis; Tomasz Szatkowski was responsible for Raman spectroscopy analysis; Allison L. Stelling and Hermann Ehrlich planned the experiment and got results development; Teofil Jesionowski coordinated all tasks in the paper, planned the experiment and got results development.

Conflicts of Interest

The authors declare no conflict of interest.

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Dyes and Pigments 134 (2016) 541e552

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Dyes and Pigments

journal homepage: www.elsevier.com/locate/dyepig

Anthocyanin dye conjugated with Hippospongia communis marine demosponge skeleton and its antiradical activity

Małgorzata Norman a, Przemysław Bartczak a, Jakub Zdarta a, Hermann Ehrlich b, * Teoﬁl Jesionowski a, a Poznan University of Technology, Faculty of Chemical Technology, Institute of Chemical Technology and Engineering, Berdychowo 4, 60965 Poznan, Poland b TU Bergakademie Freiberg, Institute of Experimental Physics, Leipziger 23, 09599 Freiberg, Germany article info abstract

Article history: A study was made of the adsorption of anthocyanin dye onto spongin-based marine demosponge Received 26 May 2016 skeletons of the species Hippospongia communis. The influence of time, dye concentration, ionic strength, Received in revised form pH and temperature on adsorption efficiency was assessed. Adsorption kinetics was analyzed using 30 July 2016 pseudo-first and pseudo-second-order models, and the pseudo-second-order model was found to Accepted 8 August 2016 represent the experimental data satisfactorily. The adsorption mechanism was verified based on Lang- Available online 9 August 2016 muir and Freundlich isotherms and the adsorption capacity (qm) reached 1413.9 mg/g. The developed sponge-hybrid materials were thoroughly analyzed using Fourier transform infrared spectroscopy (FTIR), Keywords: 13 Marine sponge cross polarization magic angle spinning nuclear magnetic resonance ( C CP MAS NMR), thermogravi- Hippospongia communis metric (TG), elemental analysis (EA) and optical and scanning electron microscopy (SEM). The antioxi- Spongin dant activity of the studied materials was also tested by estimating the ability of the dyeebiopolymer Anthocyanin system to scavenge the stable 2,2'-diphenyl-1-picrylhydrazyl free radical (DPPH). Anthocyanins were Antiradical activity adsorbed on the sponge skeleton with high efficiency, and the resulting hybrid material was able to DPPH method remove up to 95% of free radicals from solution. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Anthocyanins have the ability to absorb excess light quanta. As flavonoids, anthocyanins have electron-donating abilities [5]. The Anthocyanins are, beside betalains, carotenoids and chlorophyll, synthesis of anthocyanins is a photochemical process [8]. Different the most important group of natural dyes widespread in nature. factors affect the color and stability of these compounds, thus their Most often they are present in leaves, fruits and flowers [1e3]. molecules are highly unstable and easily susceptible to degradation Anthocyanins are plant polyphenols, derivatives of 2-phenylbenzo under the influence of pH, temperature, light, the presence of pyrylium salts (flavylium salts). The aglycone (anthocyanidin, most copigments, self-association, metallic ions, enzymes, oxygen, often pelargonidin, peonidin, cyanidin, malvidin, petunidin and ascorbic acid and sugar [9]. Anthocyanins subjected to ionizing delphinidin) moiety is glycosylated by one or more sugars and can radiation, UV or even solar radiation are susceptible to degradation, have several hydroxyl and methoxy groups. Anthocyanins differ in the flavylium cation being raised to an excited state [10]. Photo- the numbers and position of substituents and in the position of the oxidation of anthocyanins leads to an intermediate product, and sugar moieties attached to the molecule [4,5] e see Fig. 1. then, via hydrolysis of the carbon atom at position C-2, the ring is The aglyconeis the chromophore in the molecule of anthocya- opened and chalcone is produced. This compound undergoes nins; it contains delocalized electrons and is responsible for the transformation and causes degradation of the anthocyanins. color of the compound. The color depends chiefly on the chemical Due to their advantageous properties, anthocyanin compounds structure of the anthocyanins: the type of aglycone, the sub- have found a variety of applications. They are a natural coloring stituents in the flavylium cation, and the type and position of the agent for food (E163, for beverages, candies, dry mixed concen- sugar radical [6]. They absorb light in a range of 520e550 nm [7]. trates, chewing gums, yoghurts and sauces) [11], cosmetics [12], medicines, textiles and fabrics [13e16]. They are used in medicines, cosmetics and diet supplements not only as colorants, but also for their health properties, as what are called nutraceuticals and cos- * Corresponding author. E-mail address: teofi[email protected] (T. Jesionowski). meceuticals [17,18]. They also have great potential for application as http://dx.doi.org/10.1016/j.dyepig.2016.08.019 0143-7208/© 2016 Elsevier Ltd. All rights reserved. 542 M. Norman et al. / Dyes and Pigments 134 (2016) 541e552

communis were collected in the Mediterranean Sea (Tunisia) was purchased by INTIB GmbH (Germany). The first stage of preparation of the sponges involved washing them with deionized water to remove residual salt. Secondly, the samples were stored for 72 h in 3 M HCl for dissolution of possible residual calcium carbonate microparticles. Afterwards, the material was rinsed with distilled water and then dried for 24 h at 50 C in a drying oven. The adsorbate used was anthocyanin dye (ANT) isolated from the skin of grapes of the variety Vitis vinifera (GroupeGrap'Sud, France). This is a solid substance, spray-dried following isolation and used without further purification. The stock solution was prepared by dissolving an accurately weighed quantity of dye in distilled water. Experimental solutions of desired concentrations Fig. 1. Structure of cyanidin 3-glucoside. were obtained by successive dilutions with distilled water. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from sensitizers in photoelectron-chemical dye-sensitized solar cells Sigma Aldrich and used in analysis of antiradical activity. Other [19e21]. Anthocyanins are also well-known for their wide range of chemicals were of reagent grade and used as supplied. bioactive properties and health benefits. They have antioxidant, antibacterial, antiinflammatory, antiviral, antifungal [22,23], chemopreventive [24,25], anticancer and antimutagenic properties 2.2. Adsorption and desorption processes [26,27]. They have a favorable effect on vision [28], wound healing [29] and body fat metabolism [30,31]; the pigments may also The adsorption capacities of the sorbent were determined by reduce the risk of coronary heart disease through modulation of weighing 0.2 g of the sorbent into a 200 mL Erlenmeyer flask and arterial protection [9]and the functioning of the circulatory system adding 50 mL of a working solution of the dye at an appropriate [32]. It has been shown that they may prevent diseases related to concentration. After the batch experiment the samples were oxidative stress [33], inhibit DNA damage in cancer cells in vitro, filtered off under reduced pressure, and the concentration of dye in and protect against age-related decline in brain function ([11] and the aqueous solutions was determined with a UV-Vis spectropho- references therein). tometer (Jasco V75, Japan) at wavelength of 520 nm. The amount of Marine demosponges, and especially so called keratose sponges, dye adsorbed per mass unit (qt) (1) and the adsorption efficiency (E) are multicellular organisms, which typical three-dimensional pro- (2) were calculated from the equations: teinaceous skeletons have been used by humans for centuries, $ including in household applications, in medicine, and as hygienic or ðC0 À CtÞ V qt ¼ (1) artistic tools [34]. Some bioactive compounds discovered in m demosponges, having anticancer and antiinflammatory properties, have found applications in pharmacy [35e37]. Research is also C À Ct Eð%Þ¼ 0 $100% (2) being carried out into the use of specially prepared sponges in C0 wound healing. The aim is to increase absorbance of exudate and to speed up tissue regeneration [38]. Diverse skeletal structures of where C0 and Ct are the concentrations of the dye in the solution demosponges are also used in tissue engineering to produce before and after sorption respectively (mg/L), V denotes the volume effective scaffolds [39,40]. Research has shown that, with suitable of the solution (L), and m is the mass of the sponge skeleton sorbent processing, some species of sponges can be used as a permanent (g). bone substitute [41]. Kinetic experiments were performed using 1000, 2000 and To enable the wider use of anthocyanin, immobilization on a 3000 mg/L (pH ¼ 4) dye solutions at defined time intervals host is required for easy handling and improvement of stability. (1e360 min). Several attempts have been made in this area [42e45]. The Adsorption isotherms were obtained from experiments carried impregnation of marine demosponge skeletons as renewable out at pH ¼ 4 for 180 min at different initial dye concentrations source with antioxidant substances may have a positive effect in (3e10 g/L). The quantity of dye at equilibrium was calculated from certain areas of application, by maintaining free radical scavenging the equation: properties (as in the case of grape skin extract) together with $ ðC0 À CeÞ V adequate structural stability (provided by the skeleton) in a hybrid qt ¼ (3) material. m We demonstrate here for the first time a method of producing a where Ce is the concentration of dye in solution at equilibrium. new dyeemarine demosponge skeleton hybrid system that is To study the effect of pH on anthocyanin adsorption, experi- biocompatible, non-toxic and environment-friendly. This study ments were carried out at different pH values ranging from 2 to 12, focuses on the adsorption of a significant amount of natural for 60 min at a dye concentration of 500 mg/L. The pH was adjusted anthocyanin dye on H. communis marine sponge skeletons. Our using NaOH or HCl, and was regularly measured. results show that the anthocyaninis not susceptible to visible light The effect of electrolyte on dye sorption was studied by adding irradiation, oxygen or elevated temperature, and retains its color NaCl (0.01e2 M) to 50 mL of anthocyanin solution (2000 mg/L, and its beneficial properties. pH ¼ 4). After 60 min, the resultant solutions were analyzed. To assess the influence of temperature, the same procedures 2. Experimental were also performed at temperatures of 20, 30, 40 and 50 C. The efficiency of desorption of the anthocyanins from the sor- 2.1. Materials bent was measured after the adsorption process had been completed (1000e4000 mg/L, pH ¼ 4). These tests were carried out Specimens of marine demosponges of the species Hippospongia in water for 240 min. The analytical methods described above were M. Norman et al. / Dyes and Pigments 134 (2016) 541e552 543 again employed to determine the concentration of dye desorbed. equation for the scavenging percentage vs Trolox concentration.

2.3. Analysis 3. Results and discussion

To verify the effectiveness of the adsorption process, Fourier 3.1. Adsorption process transform infrared spectroscopy (FTIR) was carried out using a Bruker Vertex 70 instrument (Germany). The materials were Fig. 2 shows the quantity of anthocyanin dye adsorbed on the analyzed in the form of tablets, made by pressing a mixture of surface of the H. communis skeletons as a function of process time. anhydrous KBr (ca. 0.25 g) and 1 mg of the tested substance in a The graphs show that for all solutions there is an initial sudden special steel ring under a pressure of 10 MPa. The measurement rise in the rate and efficiency of the process. These values stabilize e À1 was carried out over a wavenumber range of 4000 400 cm at a after approximately 180 min, or earlier (from 60 min) in the case of À1 resolution of 0.5 cm . the least concentrated solutions. This indicates that the adsorption Cross Polarization Magic Angle Spinning Nuclear Magnetic process is fast at the start, gradually decreases with time and 13 Resonance ( C CP/MAS NMR) spectra of the samples were recor- eventually attains saturation when equilibrium is reached [47]. ded on a DSX spectrometer (Bruker) at 100.63 MHz. A 100 mg With an increase in the initial concentration of the solutions, the sample packed in 4 mm outer diameter ZrO2 rotors was spun quantity of anthocyanin dye adsorbed on the surface of the support (spinning speed 8 kHz). is observed to increase. This behavior may be caused by the A thermogravimetric analyzer (TG/DTA/DSC, model Jupiter STA increment in the mass gradient pressure between the adsorbate 449F3, Netzsch) was used to investigate the thermal decomposition and adsorbent, which drives the transfer of the dye from the bulk behavior of the samples. Measurements were performed in a ni- solution to the surface of the adsorbent [48]. Previously anthocy- fl 3 trogen atmosphere ( ow rate 10 cm /min) at a heating rate of anins have been adsorbed onto synthetic and natural adsorbents: e 10 C/min over a temperature range of 25 1000 C, with an initial macroporous resins were optimized to develop a simple and effi- sample weight of approximately 5 mg. cient method for industrial separation and purification of roselle The elemental contents of N, C, H and S were measured using a anthocyanins (the highest monolayer sorption capacity of a resin Vario EL Cube instrument (Elementar Analysensysteme GmbH, was 38.16 mg/g) [43]: gluten and its fractions were used as adsor- Germany). The samples were placed in the instrument and com- bents in a study that provides a basis to estimate the potential effect busted in an oxygen atmosphere. After passing through appropriate on bioavailability of anthocyanin adsorption (sorption capacities catalysts in a helium stream, the resulting gases were separated in 1.0 mg/g) [44]; and clay/HDPE particles were also tested as a po- an absorption column. tential anthocyanin adsorbent, with a sorption capacity of Samples of sponge skeletons before and after the adsorption 11.76 mg/g [45]. process were observed using a Keyence BZ 9000 microscope in light To investigate the influence of varying pH on the anthocyanins' microscopy mode. color and adsorption efficiency, absorption spectra of the dye so- The morphology and microstructure of the sponge skeleton lutions were produced (Fig. 3). support structure were studied under a Zeiss EVO40 (Germany) Anthocyanins display a red color in an acidic environment, scanning electron microscope. passing through purple to blue in a basic environment. The spectra obtained unambiguously confirm the changes taking place. There is 2.3.1. Determination of antioxidant activity with the DPPH radical an increase in the maximum wavelength, responsible for the scavenging method perception of blue color (600e630 nm) compared with the previ- Antioxidant activity was determined by a modified version of ously observed purple (up to 600 nm) and red (from 500 nm). The the Brand-Williams method [46] using the 2,2-diphenyl-1- mechanism of the change in color results from the instability of the picrylhydrazyl (DPPH) radical. With this method it is possible to described compounds. The red flavylium cation (AHþ) affects determine the antiradical activity of an antioxidant by measure- structural transformations on an increase in pH leading to colorless ment of the decrease in absorbance of DPPH. The measurement was hemiketal carbinol pseudobase (B), purple neutral quinoidal base made as follows: 0.5 mL of the DPPH solution (50 mg/100 mL) was diluted in 4.5 mL of methanol, and 1 mg of the tested material was added (or 1 mL in the case of aqueous anthocyanin solution). H. communis skeletons and hybrid material obtained from anthocyanin solutions (0.2 g of sponge skeleton, 50 mL of dye solution in the range 1e10 g/L) were the tested substances. The mixture was shaken in the dark for 30 min. The change in absorbance (A)was measured at 514 nm against a blank (without tested materials; A0) with a spectrophotometer. Methanol was used as a reference. Each measurement was repeated three times, and the mean absorbance was calculated for each tested material. The efficiency of the process (the antioxidant activity; AA) was computed from the equation:

A0 À A AAð%Þ ¼ $100 (4) A0 Moreover, the scavenging activity of the samples was compared with the activity of Trolox. The measurements was made in the same way as in case of sponges skeleton with anthocyanins, but instead of 1 mg of sample 1 ml of Trolox methanolic solution (at different concentration) was added. The Trolox equivalent antiox- Fig. 2. Time-dependence of effectiveness of adsorption of anthocyanin onto idant capacity (TEAC) was calculated from the linear regression H. communis skeletons from dye solution at different concentrations. 544 M. Norman et al. / Dyes and Pigments 134 (2016) 541e552

Fig. 3. Different forms of anthocyanins and absorption spectra of anthocyanin solution at room temperature at varying pH.

(N) and to blue anionic quinoidal base (A) [49]. Equilibrium be- are given in Table 1. tween AHþ and B is attained at a pH of 2.6. Equilibrium for the Theoretically, if there is attractive electrostatic interaction be- cation (AHþ) and base (N) occurs at a pH of 4.25. Pale yellow tween adsorbate and adsorbent, then an increase in ionic strength chalcone (C) is produced by spontaneous reactions and further should cause a decrease in the adsorptive capacity of the adsorbent. transformations of the pseudobase [1,50]. The existence of antho- The converse is found in case of repulsive interaction; increasing cyanins in different forms depending on pH is shown in Fig. 3. ionic strength then causes an increase in adsorptive capacity. An The effect of pH on the efficiency of the adsorption process was increase in ionic strength causes a reduction in the thickness of the also studied. The process was carried out from a solution with electrical double layer, and thus a reduction in repulsive in- concentration 500 mg/L, for 60 min, over the pH range 2e12. The teractions. The aggregation of molecules of the dye in the solution results showed that the optimum environment for a solution of is affected by intermolecular interactions such as van der Waals anthocyanin dye has a pH of 4 (this is a neutral pH for anthocyanin forces, ion-ion interactions and dipole-dipole interactions. These solutions, without adding any HCl or NaOH). The adsorption effi- forces are increased when NaCl is added to the dye solution. Hence ciency here was 72.5%, compared with approximately 50% in basic increased adsorption capacity may be a result of aggregation of the and 40% in acidic environments. As mentioned above, with an indye molecules caused by the action of ions of the salt e that is, ions crease in pH anthocyanins pass from the form of a cation (AHþ) of the salt force the dye molecules to accumulate, thus increasing through a neutral form (N) to a base (A). In an acidic environment the extent of sorption on the surface of the adsorbent [52]. This can the functional groups of the amino acids forming spongin in the be observed on addition of even a small quantity of NaCl, when the þ þ sponge skeletons are protonated (eNH3 , eCOOH2 ). An increase in quantity of dye adsorbed increases significantly from around 131 to alkalinity may cause the formation of eNHÀ and eCOOÀ groups on 214 mg/g. A doubling of the quantity of dye and of the process ef- the support surface, whereas the dye exists as blue anionic qui- ficiency to more than 54% was obtained at a concentration of 1 M noidal base (A). The existence of these forms of adsorbent and NaCl. It was found that as the quantity of NaCl increases there is an adsorbate makes mutual interaction difficult [51]. The mechanism increase in the efficiency of the adsorption process, and thus in the of adsorption may thus be partially explained by electrostatic quantity of anthocyanin dye retained on the surface of the sponge interaction. Lower process efficiency may be caused by mutual skeletons. This observation again indicates the presence of elec- repulsion of charges in a strongly acidic or strongly alkaline envi- trostatic forces between the support and the dye. As reported in ronment. The highest efficiency is a result of the existence of the Ref. [53], hydrophobic attraction was also found to increase with forms (AHþ) and (N) in equilibrium at pH ¼ 4.25; in such an increasing ionic strength. environment it is possible for hydrogen bonds to form between the An analysis was also made of the effect of temperature on the adsorbent and adsorbate. The presence of hydrogen bonds is also adsorption process. The experiment was carried out using an confirmed by the results of spectroscopic analysis described below. aqueous solution of dye at a concentration of 2000 mg/L, in an The proposed mechanism of interaction is illustrated in Fig. 4. environment with a pH of 4, over 60 min. This was done at tem- An investigation was also made of the influence of ionic strength peratures in the range 20e50 C, with an increment of 10 C be- on the dye adsorption process in a reactive environment with tween measurements. The results are given in Table 1. neutral pH, concentration 2000 mg/Land time 60 min. The results The results show that an increase in temperature is favorable to M. Norman et al. / Dyes and Pigments 134 (2016) 541e552 545

Fig. 4. Mechanism of interaction between anthocyanin and H. communis skeletons at different pH values.

Table 1 To investigate the kinetics of the process of adsorption of dye on Results obtained for adsorption carried out at different the support, pseudo-ﬁrst-order (PFO) and pseudo-second-order concentrations of electrolyte (NaCl) and at different tem- (PSO) models were used. The equations used to calculate the ki- peratures (adsorption process conditions: 2000 mg/L, 60 min, pH ¼ 4). netic parameters of the process, and the computed results, are given in Table 2. The experimental quantity of adsorbed dye at NaCl (mol/L) qe (mg/g) equilibrium qexp and the adsorption rate k were calculated exper- 0 131.1 imentally from the relationship between log(qe À qt) and t for the 0.01 214.1 pseudo-ﬁrst-order model (Fig. 5a) and from that between t/qt and t 0.1 226.3 1 270.8 for the pseudo-second-order model (Fig. 5b). The adsorption pro- 2 281.0 cess was carried out from dye solutions with concentrations of

1000, 2000 and 3000 mg/L. For each concentration of solution, the Temperature ( C) qe (mg/g) adsorption efficiency was calculated for times in the range 20 131.1 1e360 min. 30 198.1 fi 2 40 241.5 The correlation coef cient r for the PFO reaction model ob- 50 240.0 tained for adsorption of the anthocyanins on sponge skeletons at dye solution concentrations of 1000, 2000 and 3000 mg/L lies in the range 0.853e0.958, while for the PSO reaction model the value the adsorption process. The optimum temperature for adsorption on sponge skeletons was found to be 40 C, since a further increase does not produce significant changes. An increase in the tempera- Table 2 ture leads to an increase in dye adsorption capacity (qt), which Effect of dye solution concentration on kinetic parameters of adsorption of antho- suggests a kinetically controlled and endothermic process. This cyanins on H. communis skeletons. may be a result of increase in the mobility of the dye with Parameters Dye solution concentration (mg/L) increasing temperature. The increase in temperature can offer 1000 2000 3000 sufficient energy to undergo the interaction between the dye q (mg/g) 129.861 245.889 341.167 molecules and active sites on the surface of sorbent [54], and thus exp increase the adsorption efficiency. Moreover, the decrease in the Pseudo-first-order * k1 $ thickness of the boundary layer surrounding the adsorbent causes a logðqe À qt Þ¼logðqeÞÀ 2;303 t decrease in the mass transfer resistance of the adsorbate [55]. r2 0.853 0.959 0.958

k1 (1/min) 0.011 0.014 0.018 qcal (mg/g) 45.44 195.92 183.54 3.2. Adsorption kinetics and isotherms Pseudo-second-order t 1 1 $ ** ¼ 2 þ t qt k q qe In order to investigate the mechanism of sorption and potential 2 e 2 rate-controlling steps such as mass transport and chemical reaction r 0.997 0.992 0.999 k (g/mg$min) 0.0013 0.0002 0.0004 processes, kinetic models have been used to analyze experimental 2 qcal (mg/g) 129.34 259.45 347.44 data [56]. The controlling mechanisms of the adsorption process, * ** , q and q are the adsorption capacities at time t and at equilibrium, respectively; such as chemical reaction, diffusion control and mass transfer co- t e q and q are the experimental and calculated amounts of adsorbed dye on the fi exp cal ef cient, are used to determine kinetic models. Linear regression support at equilibrium; k1 and k2 are the rate constants of pseudo-first-order and was used to determine the best-fitting kinetic rate equation. pseudo-second-order adsorption; t is the contact time. 546 M. Norman et al. / Dyes and Pigments 134 (2016) 541e552

Fig. 5. Plots of pseudo-first-order (a) and pseudo-second-order (b) kinetic models for adsorption of anthocyanins from solutions of different concentrations on H. communis skeletons. of the correlation coefficient is in the range 0.992e0.999. Similarly, the quantity of dye adsorbed at equilibrium, qcal, computed from the PFO model deviates significantly from the experimental values qexp. This means that the PFO reaction model does not give a direct correspondence to the experimental data. A much greater similarity to the theoretical parameters computed from the model, qcal,is obtained in the case of the PSO model. Equilibrium isotherm equations are used to describe experimental sorption data. The equation parameters and the assumptions of these models may supply some insight into the mechanism of this process as well as the surface properties and affinity of the adsorbent. The Langmuir isotherm model assumes that the adsorption process takes place in a monolayer and that each adsorption site is homogeneous. Specifically, adsorption takes place when a free adsorbate molecule collides with an unoccupied adsorption site. The Freundlich isotherm model does not have much limitations, and assumes neither homogeneous site energies nor limited levels of sorption [57,58]. In the sorption process on Fig. 6. Fitting of the Langmuir and Freundlich isotherm models to equilibrium results sorbents of natural origin, the saturation limit of a support is for anthocyanin dye adsorbed onto H. communis skeletons. affected by several factors such as the number of sites in the biosorbent material, the accessibility of the sites, the chemical state of Table 3 fi the sites and the af nity between the adsorbate and support [59]. Parameters of isotherm models of dye adsorption on H. communis skeletons. Fig. 6 shows the equilibrium adsorption isotherm and fitting of the Isotherm model Langmuir Freundlich models to experimental data. According to the calculations 2 2 (Table 3), the correlation to sorption isotherms is greatest in the Parameters R qm (mg/g) b (L/mg) R KF (mg/g) n case of a Freundlich-type isotherm. The very large quantity of dye 0.863 1413.9 0.001 0.986 1.4 1.78 adsorbed on the sponge skeletons is more likely if the adsorption is of multilayer type. the more is available to be removed from that support, and this im- pacts the efficiency of desorption of the dye from the support surface. 3.3. Desorption process Nevertheless, the desorption efficiency is still low, as only a small amount of dye could be desorbed from the support. It appeared that For the purpose of carrying out a process of desorption of dye from the adsorption of anthocyanins on H. communis skeletons was highly marine sponges, the substrate was first adsorbed on the surface of the favorable, tending to be weakly reversible and indicating that the dye support. The supportedye system was obtained from an aqueous is relatively strongly bound to the support. Different mechanisms solution of dye with a concentration of 1000, 2000, 3000 or 4000 mg/ may be responsible for the interaction between adsorbent and L in a time of 1 h. The next stage involved desorption of the antho- adsorbate molecules. The results of the investigations of the influ- cyanin dye from the surface of the biopolymer in the presence of ence of pH and ionic strength on adsorption efficiency indicate the distilled water. The efficiency of desorptionwas lowest for the system presence of electrostatic interaction. obtained from a solution of concentration 1000 mg/L (13%), and highest for 4000 mg/L (27%). Intermediate values were obtained for concentrations of 2000 and 3000 mg/L (14% and 22% respectively). 3.4. FTIR There is a simple correlation between adsorption and desorption efficiency. The more dye is adsorbed on the marine sponge skeletons, Fig. 7 shows the FTIR spectra of H. communis skeletons, M. Norman et al. / Dyes and Pigments 134 (2016) 541e552 547

Fig. 7. FTIR analysis of (a) H. communis skeleton, dye and selected samples (1. 9000 mg/L, 1 h, pH ¼ 4; 2. 8000 mg/L, 1 h, pH ¼ 4; 3. 7000 mg/L, 1 h, pH ¼ 4); (b) expanded spectra in the wavenumber range 1800e500 cmÀ1.

anthocyanin dye and dyeeskeleton systems obtained in a time of signals related to functional groups present in the H. communis 1 h at initial pH from dye solutions with concentrations of 7000, spongin and anthocyanins, which confirm effective adsorption of 8000 and 9000 mg/L (samples 1, 2 and 3). The presence of similar the dye on the sponge skeletal protein. Moreover, the presence of signals on the spectra for the sponges and the dye results from the the dye causes signal values (peak maxima) to change, especially in presence of the same functional groups within their structures. the ranges 1035e110 0 cm À1, 1500e1750 cmÀ1 and below H. communis skeletons, built from spongin, contain functional 1000 cmÀ1, in comparison with the sponge skeleton, as can be seen groups characteristic for proteins. This biopolymer still has un- in the more detailed spectra (Fig. 6 b). Because of the clearly visible known chemical structure, but the amino acid composition of deformations of the eOH bands in the spectra of dyeebiopolymer, spongin is similar to vertebrate collagen, with a high percentage of we suggest that these groups are interacting with the H. communis glycosylated hydroxylysine, aspartic acid, and glutamic acid [60]. skeletons. It is also should be noted that intensity of the signal at Proteins have broad, strong bands which, due to considerable 1730 cmÀ1 in the spectra of the hybrid materials related to the C]O overlap, are difficult to differentiate; for example, those in the range bonds vibrations at the ANT dye after adsorption process is much 3600e3100 cmÀ1 may come from OeHorNeH stretching vibra- lower. This phenomenon can be explained by the fact that in the tions. The signals in the range 2950e2850 cmÀ1 are related to the reaction environment partially protonization of these groups is stretching vibrations of CeHineCH3 and eCH2. The broad band in possible and the creation of the hydrogen bonds is favorable, which the range 1710e1640 cmÀ1 comes from stretching vibrations of the increase affinity of the dye particles to the marine sponge skeletons. carbonyl group C]O. The spectra of all proteins exhibit absorption Moreover, in the pH around 4, transformation of the dye molecules bands due to their characteristic amide group, COeNH; the char- into the different tatutomeric form (not included C]O bonds) is acteristic bands of the amide group of protein chains are similar to highly possible [50]. Additionally slightly red-shifts are observed in those of ordinary secondary amides, in this case at 1540 cmÀ1. the case of the signals related to the vibrations of C]C aromatic Signals confirming the presence of aromatic rings in the sponge bonds. This fact is explained by the changes in the chemical envi- À1 structure are observed at 1450 cm (CAr]CAr). The band at ronment of the dye due to the adsorption process, as reported wavenumber 1390 cmÀ1 is associated with deformational vibra- earlier by Vankar and Shukla [62]. tions of eCH3. Stretching vibrations of CeO are represented at 1230 (CeOeC) and at 1070 and 1020 cmÀ1(CeOH). The signal at 3.5. 13C CP/MAS NMR 660 cmÀ1 can be attributed to O]CeN or OH bending vibrations. The bands of ANT are labeled according to [61]. The spectrum of The results of chemical structure analyses performed using NMR the anthocyanin dye has a strong band with a maximum at show strong evidence for the effective adsorption of anthocyanin 3405 cmÀ1 corresponding to OeH stretching vibrations. Peaks at on the selected H. communis skeletons. 13C CP/MAS NMR spectra of 2934 and 2886 cmÀ1 reflect stretching vibrations of CeH bonds in the dye, adsorbent and obtained hybrid material are shown in Fig. 8 À1 eCH2and eCH3 groups. The signal at 1732 cm is associated with a, b and c, respectively. A detailed characterization of anthocyanin the presence of the C]O functional group (stretching vibrations of 13C NMR chemical shifts appears in Refs. [4] and [63]. The broad ester groups). The peaks at 1076 and 1027 cmÀ1 correspond to signal at 69.88 and 60.69 ppm comes from sugar moiety carbons stretching vibrations of CeOeC bonds. Stretching vibrations of CeO (the value can differ depending on the type of sugar). Although bonds also give rise to a signal with a maximum at 1117 cmÀ1. The there is no chemical shifts characteristic for ANT below 60 ppm, on bands at 1628 and 1516 cmÀ1 correspond to C]C stretching vi- our spectrum some appear. The additional signals (at around brations, confirming the aromatic ring in the anthocyanin struc- 30 ppm, for example) may originate from components of the dye À1 ture. The signal at 1328 cm comes from eCH2 groups, and those other than anthocyanins. The less intense signals occurring in the below 1000 cmÀ1 from the sugar moiety in the anthocyanin range 90e175 ppm come from the aglycone molecule (anthocya- molecule. nidin). In the case of H. communis skeletons the peak observed at The spectra of the products of the sorption process contain d ¼ 178.2 ppm corresponds to the carbon of a carboxyl group (C] 548 M. Norman et al. / Dyes and Pigments 134 (2016) 541e552

Fig. 8. 13C CP/MAS NMR spectra of (a) dye, (b) H. communis skeleton, and (c) hybrid material obtained from 5000 mg/L dye solution (pH ¼ 4, 1 h).

O), and that at 150.75 ppm (much less intense) to aromatic carbons. 3.6. Thermal stability The results indicate the presence of aliphatic carbon (saturated alkanes), carbon bonded to nitrogen (CeNR2) and to oxygen (CeOH, The results of thermal analysis are presented in Fig. 9. CeOR) with signals in the 20e80 ppm range. The 13C NMR spectra Anthocyanins are susceptible to degradation under the inﬂu- of the hybrid material showed similarities with the spectra of the ence of many factors, including temperature. From the TG curve for marine sponge skeleton, including signals corresponding to the ANT it can be seen that a slight mass loss (less than 1%) begins at ANT points. Additional signals, especially above 100 ppm, become 30 C, and from 130 to 650 C proceeds more rapidly but steadily. narrow and hence more visible. Furthermore, several line splittings Heating to around 100 C accelerates the processes of oxidative are visible, especially at 169.28 and 168.09 ppm, and also at 71.76, polymerization and color change of the anthocyanins [8]. The 69.49 and 68.10 ppm. Moreover, after adsorption of ANT on the brown color of these compounds may be caused by hydrolyzation H. communis skeleton the 13C NMR signal was shifted. This indicates of the glycoside structure of the C ring, resulting in the production chemical interaction between the dye and adsorbate. of chalcones [64]. The chalcone then undergoes transformation and M. Norman et al. / Dyes and Pigments 134 (2016) 541e552 549

is a residue from the extraction process. In the case of nitrogen, there is a signiﬁcant difference in the percentage content between the support and the dye. The sponge skeleton has more than 15% of that element in its protein structure, while the dye contains just 1.4%. Unfortunately, the limitations of this method of analysis mean that it is not possible to determine the contents of other elements. Nevertheless, H. communis skeletons consist also of several other different elements (O, I, Al, Cl, Si). For the tested samples the quantity of nitrogen declines with increasing dye solution concentration. Combining anthocyanin, having a low content of this element, with a support having a high content results in propor- tional reduction of the nitrogen content in the samples, and conversely in the case of sulfur. These results also indirectly conﬁrm the effectiveness of the adsorption process.

3.8. Microscopy observations

Several optical microscopy (Fig. 10) and SEM (Fig. 11) images were taken of H. communis skeletons before and after adsorption of Fig. 9. Thermal stability curve of H. communis skeletons, dye and hybrid material anthocyanins. obtained from 7000 mg/L (1) and 10,000 mg/L (2) dye solution, at pH ¼ 4over1h. The images from the optical microscope clearly demonstrate that the adsorption process was successful. The fibers of the skeleton were uniformly covered with the dye. These observations are causes degradation of the anthocyanins. Hydrolyzation of the supported by the results of SEM analysis. SEM microphotographs of glycoside involves detachment of the sugar moiety, followed by H. communis after acidic treatment confirmed the characteristic decomposition of the aglycone into phloroglucinaldehyde (cyani- organization of the sponge fibers. Spongin fibers may range in din, pelargonidin), -hydroxybenzoic acid (pelargonidin), and pro- thickness from a few to about 10e15 mm, and consist of bundles of tocatechuic acid (cyanidin), the residues of the A- and B-rings fibrils/microfibrils ([67] and references therein). This is referred to respectively [65]. as the hierarchical, multilevel organization of spongin. After the The thermal destruction of the marine sponge skeletons studied adsorption process the microfibrils are no longer visible because a takes place in three stages, and may be associated with water loss layer of dye fully covers the spongin fibers. and decomposition of the organic phase [66]. Both analyzed samples exhibit an increase in thermal stability 3.9. Determination of antioxidant activity with the DPPH radical up to 200 C in comparison with ANT. Moreover the lower mass scavenging method loss, compared to H. communis skeleton, in whole range of temperature is observed. The effect is better visible for product ob- The DPPH radical scavenging method is widely used for rapid tained from higher concentration of the dye solution. This could be estimation of the antioxidant activity of various compounds. DPPH an indirect proof of effectiveness of the adsorption. Additionally, at is a stable free radical which has a very broad visible absorption 1000 C still 32 and 37% of mass is present (respectively for sample band with a maximum at 514 nm in MeOH. When an antioxidant is 1 and 2), which is the result of carbonization. added to DPPH, the free electron is paired up (by acceptance of an electron or hydrogen) and a stable DPPH-H molecule is formed [68]. 3.7. Elemental analysis This results in a color change from purple to yellow, which is measured as a decrease in absorbance. Anthocyanins are poly- In order to make a thorough analysis of the composition of the phenols, and the antioxidant activity of these compounds may be tested materials, a series of elemental analyses were performed to explained by their capacity to act as reducing agents by donating a determine the contents of carbon, nitrogen, hydrogen and sulfur. hydrogen, by quenching singlet oxygen, or by acting as chelators Table 4 contains results for the marine sponge skeletons, the dye, [69]. The number of hydroxyl and methoxy groups in the B-ring, the and three dyeesupport systems obtained from solutions of initial aglycone moiety and the glycoside forms and the acylation by concentration 9000, 6000 and 2000 mg/L (60 min, pH ¼ 4). phenolic acids are related to the antioxidant capacity of these Both marine sponge skeleton and the dye are organic sub- compounds [70,71]. After donating a proton, one of the possible stances, hence the high content of carbon and (relatively) paths for polyphenols having a 1,2-dihydroxysubstitution on their hydrogen. Sulfur is a component of cysteine, an amino acid, which B-ring is conversion into semiquinones, which are stabilized by a also occurs in the structure of sponging [67]; in the case of the dye it combination of electronic and intramolecular hydrogen bonding effects. Other oxidation pathways lead to the formation of mixtures of dimers and oxidized dimers, C-ring cleavage and/or B-ring Table 4 elimination [72,73]. Elemental analysis of marine sponge skeletons, dye and selected samples (1. The antioxidant capacity of H. communis skeletons, anthocyanin 9000 mg/L, 1 h, pH ¼ 4; 2. 6000 mg/L, 1 h, pH ¼ 4; 3. 2000 mg/L, 1 h, pH ¼ 4). solution and selected hybrid was tested. The dyeeskeleton systems Elemental content (%) were obtained from anthocyanin solutions of different concentra- NC HStions (3e10 g/L) for 1 h at pH ¼ 4.

H. communis skeleton 15.25 42.44 5.75 0.71 The concentration of DPPH radicals following the test decreases Anthocyanin 1.41 44.19 4.68 1.46 with increasing quantity of dye on the surface of the sample, and Sample 1 11.96 43.79 5.44 1.20 hence the antioxidant power rises. This decrease in concentration Sample 2 12.80 43.50 5.48 1.09 implies a color loss, which indicates that all of the tested hybrid Sample 3 14.56 44.19 5.54 0.92 materials had scavenged the free radical. Fig. 12 illustrates the 550 M. Norman et al. / Dyes and Pigments 134 (2016) 541e552

Fig. 10. Light microscopy images of H. communis skeletons before (a, b), and after the adsorption process (c, d), at different magniﬁcations.

Fig. 11. SEM images of H. communis skeletal fibre before (a) and after (b, c) the adsorption process. results obtained for the absorbance of the tested solutions. The adsorbed in 1 mg of dye-sponge hybrid obtained from 10 g/L so- efficiency of the process ranges from 95.8% (0.0443 mg/mL of lution, as show the isotherms calculations. The results indicate that Trolox) for the system obtained from a dye solution of concentra- even after adsorption the dye preserves its beneficial properties. tion 10 g/L to 55.9% (0.0238 mg/mL of Trolox) for the system prepared from 3 g/L anthocyanin solution. Measurements of 4. Conclusions antiradical power were also made for pure H. communis skeletons, but no activity was observed in this case. The experiments for By means of adsorption batch experiments we have created an aqueous solutions of anthocyanin give results similar to those ob- environmentally friendly and cost-effective dyeebiopolymer tained for sponge skeleton-ANT system: 0.04221 mg/ml of Trolox hybrid material with antioxidant properties. The high antioxidant for 1000 mg/L of ANT solution. The amount of ANT in 1 mL of activity of the anthocyanins is attributed to the specific structure of 1000 mg/L dye solution corresponds with amount of anthocyanins the dye. The adsorption rate was extremely high in the initial M. Norman et al. / Dyes and Pigments 134 (2016) 541e552 551

Fig. 12. Changes in absorbance of DPPH solutions after reaction with dyeemarine sponge skeleton hybrid material (obtained from 3 to 10 g/L dye solution). contact time, and no significant changes were observed after Plant Physiol Biochem 2010;48:1015e9. equilibrium was reached. Similarly, the efficiency was constant [5] Janeiro P, Brett AMO. Redox behavior of anthocyanins present in Vitis vinifera L. Electroanal 2007;19:1779e86. after equilibrium due to the slow pore diffusion or saturation of the [6] Heredia FJ, Francia-Aricha EM, Rivas-Gonzalo JC, Vicario IM, Santos-Buelga C. adsorbent, and the adsorption percentage was stable after longer Chromatic characterisation of anthocyanins from red grapes: 1. pH effect. e times. The Freundlich isotherm and PSO model better describe the Food Chem 1998;63:491 8. [7] Kumara NTRN, Ekanayake P, Lim A, Liew LYC, Iskandar M, Ming LC, et al. experimental data. Ionic strength and pH are two important factors Layered co-sensitization for enhancement of conversion efficiency of natural 13 affecting dye adsorption. C CP/MAS NMR, TG and elemental dye sensitized solar cells. J Alloy Compd 2013;581:186e91. analysis results indirectly confirmed the successful adsorption [8] Pia˛tkowska E, Kopec A, Leszczynska T. Antocyjany-charakterystyka, wyste˛powanie i oddziaływanie na organizm człowieka. Zywn Nauk Technol Ja process. Analysis suggests that H. communis skeletons can also 2011;4:24e35. interact with dye molecules via hydrogen bonding. Precise verifi- [9] Cavalcanti RN, Santos DT, Meireles MA. Non-thermal stabilization mecha- cation of the mechanism will be the subject of further research. nisms of anthocyanins in model and food systems: an overview. Food Res Int 2011;44:499e509. [10] Lee SV, Hadi AN, Zainal Abidin ZHZ, Mazni NA. Thermal and UV degradation of Acknowledgments roselle anthocyanin extract and its mixtures with poly(vinyl alcohol) in different acid. Pigment Resin Technol 2015;44:109e15. fi [11] Chandrasekhar J, Madhusudhan MC, Raghavarao KSMS. Extraction of antho- This work was nancially supported by Poznan University of cyanins from red cabbage and purification using adsorption. Food Bioprod Technology research grant no. 03/32/DSMK/0610. Process 2012;90:615e23. [12] Boga C, Delpivo C, Ballarin B, Morigi M, Galli S, Micheletti G, et al. Investigation on the dyeing power of some organic natural compounds for a green References approach to hair dyeing. Dyes Pigments 2013;97:9e18. [13] Mansour R, Ezzili B, Farouk M. Dyeing properties of wool fabrics dyed with [1] Stintzing FC, Carle R. Functional properties of anthocyanins and betalains in Vitis Vinifera L. (black grenache) leaves extract. Fibers Polym 2013;14:786e92. plants, food, and in human nutrition. Trends Food Sci Tech 2004;15:19e38. [14] Pawlak K, Puchalska M, Miszczak A, Rosłoniec E, Jarosz M. Blue natural organic [2] Tanaka Y, Sasaki N, Ohmiya A. Biosynthesis of plant pigments: anthocyanins, dyestuffs e from textile dyeing to mural painting. Separation and character- betalains and carotenoids. Plant J 2008;54:733e49. ization of coloring matters present in elderberry, logwood and indigo. J Mass [3] Tatsuzawa F, Toki K, Saito N, Shinoda K, Shigihara A, Honda T. Anthocyanin Spectrom 2006;41:613e22. occurrence in the root peels, petioles and flowers of red radish (Raphanus [15] Shukla D, Vankar PS. Natural dyeing with black carrot: new source for newer sativus L.). Dyes Pigments 2008;79:83e8. shades on silk. J Nat Fibers 2013;10:207e18. [4] Srivastava J, Vankar PS. Canna indica flower: new source of anthocyanins. [16] Shanker R, Vankar PS. Dyeing cotton, wool and silk with Hibiscus mutabilis 552 M. Norman et al. / Dyes and Pigments 134 (2016) 541e552

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Article Sodium Copper Chlorophyllin Immobilization onto Hippospongia communis Marine Demosponge Skeleton and Its Antibacterial Activity

Małgorzata Norman 1, Przemysław Bartczak 1, Jakub Zdarta 1, Wiktor Tomala 1, Barbara Zura˙ ´nska 1, Anna Dobrowolska 2, Adam Piasecki 3, Katarzyna Czaczyk 2, Hermann Ehrlich 4 and Teoﬁl Jesionowski 1,*

1 Institute of Chemical Technology and Engineering, Faculty of Chemical Technology, Poznan University of Technology, Berdychowo 4, 60965 Poznan, Poland; [email protected] (M.N.); [email protected] (P.B.); [email protected] (J.Z.); [email protected] (W.T.); [email protected] (B.Z.)˙ 2 Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, 60627 Poznan, Poland; [email protected] (A.D.); [email protected] (K.C.) 3 Institute of Materials Science and Engineering, Faculty of Mechanical Engineering and Management, Poznan University of Technology, Jana Pawla II 24, 60965 Poznan, Poland; [email protected] 4 Institute of Experimental Physics, TU Bergakademie Freiberg, Leipziger 23, 09599 Freiberg, Germany; [email protected] * Correspondence: teoﬁ[email protected]; Tel.: +48-61-665-3720

Academic Editor: Iolanda Francolini Received: 28 July 2016; Accepted: 9 September 2016; Published: 27 September 2016

Abstract: In this study, Hippospongia communis marine demosponge skeleton was used as an adsorbent for sodium copper chlorophyllin (SCC). Obtained results indicate the high sorption capacity of this biomaterial with respect to SCC. Batch experiments were performed under different conditions and kinetic and isotherms properties were investigated. Acidic pH and the addition of sodium chloride increased SCC adsorption. The experimental data were well described by a pseudo-second order kinetic model. Equilibrium adsorption isotherms were determined and the experimental data were analyzed using both Langmuir and Freundlich isotherms. The effectiveness of the process was conﬁrmed by 13C Cross Polarization Magic Angle Spinning Nuclear Magnetic Resonance (13C CP/MAS NMR), Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDS) and thermogravimetric analysis (TG). This novel SCC-sponge-based functional hybrid material was found to exhibit antimicrobial activity against the gram-positive bacterium Staphylococcus aureus.

Keywords: marine sponge; Hippospongia communis; chlorophyllin; hybrid materials; antibacterial activity

1. Introduction The utilization of materials of natural origin like both structural polysaccharides (chitin, cellulose) and structural proteins (collagen, keratin, silk, spongin) has been gaining increasing scientiﬁc attention in recent years. Key features contributing to the popularity of these renewable biomaterials include biodegradability, ecological safety, low cost, renewability, and high compatibility with the environment. Chlorophyllin (see Figure 1b), a chlorophyll derivative, is obtained as a product of solvent extraction of grass, lucerne, nettle and other plant material. Further, saponiﬁcation removes the methyl and phytol from the chlorophyll molecule, and may partially cleave the pentenyl ring (depending on the degree of hydrolysis [1], the cyclopentenyl ring may be cleaved with the resultant production of a third carboxyl function). This procedure leads to a complex mixture of compounds [2–4]. In chemical

Int. J. Mol. Sci. 2016, 17, 1564; doi:10.3390/ijms17101564 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2 of

methyl and phytol from the chlorophyll molecule nd may partially cleave the pentenyl ring (depending on the degree of hydrolysis [1], the cyclopentenyl ring may be cleaved with the resultant Int.production J. Mol. Sci. 2016of a, 17third, 1564 carboxyl function) This procedure leads to a complex mixture of compounds2 of 17 [2 ]. In chemical terms, this is a m crocyclic molecule consisting of four pyrrole rings connected by terms,methylene this bridges, is a macrocyclic with met molecule l ion inside consisting (Figure 1). of The four most pyrrole common rings form connected is the sodium–copper by methylene bridges,derivative with (chlorophyllin a metal ion inside sodium (Figure 1copper). The mosts lt common or sodium form is thecopper sodium–copper chlorophyllin–SCC) derivative C H CuN Na O [5]. The replacement of the centr l magnesium ion with copper produces a more (chlorophyllin sodium copper salt or sodium copper chlorophyllin–SCC), C34H31CuN4Na3O6 [5]. Thestable replacement complex with ofthe greater central tinctorial magnesium strength. ion As with well copper s copper, produces divalent a more cations stable such complex s iron with and greaterzinc may tinctorial be used strength. [6–9]. As well as copper, divalent cations such as iron and zinc may be used [6–9].

FigureFigure 1.1. Chemical structure ofof chlorophyllchlorophyll ((a)) andand chlorophyllinchlorophyllin ((bb).).

UnlikeUnlike chlorophyll,chlorophyll, SCCSCC isis waterwater soluble,soluble, duedue toto replacementreplacement ofof thethe phytolphytol withwith aa carboxylatecarboxylate groupgroup [[10]. ] Moreover, SCC is very slightly soluble in lower alcohols, lcohols, ketones and diethyl ether. However,However, itit isis insolubleinsoluble inin chloroalkanes,chloroalk nes hydrocarbonshydrocarbons andand fixedfixed oils oils [[3]. ] ChlorophyllinsChlorophyllins haveh ve greatgre t potentialpotential for biomedicbiomedical l application. application. SCC, SCC, considered considered as asnontoxic, nontoxic, h hass been been used used historically historically in the in thetreatment treatment of ofseveral several human human conditions conditions [1 [11,12 ]]. Nevertheless Nevertheless, more more recent recent studies studies have shownshown tumor-enhancingtumor-enh ncing andand genotoxicgenotoxic effectseffects ofof thethe complexcomplex [13[1]. ] ChlorophyllinChlorophyllin exhibitsexhibits antioxidant,antioxidant antimutagenic,ntimutagenic, andand anticarcinogenic nticarcinogenic propertiesproperties inin severalseveral models.models. It is known to bindbind toto planar planar compoundscompounds suchsuch asas heterocyclicheterocyclic amines mines [[14,1514,15],], dioxindioxin [[16],16], aflatoxinaflatoxin [[1717,18 18],], andand benzo[benzo[a]pyrene [[19]19]. TheThe antimutagenicantimutagenic activity ctivity of of chlorophyllin chlorophyllin comes comes also also from from the scavengingthe scavenging of free of radicals free r dicand lsactive and oxygenactive oxygen species, species, and suppression and suppression of or interference of or interference with metabolic with activation met bolic byactivation a specific by cytochrome specific (P-450)cytochrome and other (P-4 metabolizing ) and other metabolizing enzymes [20,21 enzymes]. Other [20,21] bioactivities Other are bioactivities also attributed are also to chlorophyllin, attributed to suchchlorophyllin as immunomodulatory such as immunomodulatory and antiapoptotic effects and an [22ti], poptotic antioxidant effects activity [22] against ntioxidant oxidative activity stress org radiation-generated inst oxidative stress reactiveor radiation-generated oxygen species reacti [23ve,24 oxygen], and antibacterialspecies [23,24], effects and antib [5,25– cterial27]. SCC effects is used[5,25–27]. as a dietarySCC is used supplement, s diet in ry food,supplement drugs and in food cosmetics drugs [28 nd],in cosmetics textile dye [ ] [1 in,29 textile], as an dye internal [ ] deodorizeras an internal [30 deodorizer] and as a natural [ ] and wound as n healer tur l [wound31]. healer [ ] ThereThere areare fewfew reportsreports toto date date concerningconcerning thethe adsorptionadsorption andand furtherfurther useuse ofof materialmateri l composedcomposed ofof SCCSCC withwith aa support.support. Chlorophyllin with TiO 2 isis used as s a photosensitizerphotosensitizer [[32 2]] inin artificial rtifici l photosynthesis,photosynthesis, chlorophyllin-chitosanchlorophyllin-chitosan as a traptrap forfor polycyclicpolycyclic mutagenicmut genic compoundscompounds [[33], ] andand heterocyclicheterocyclic aminesamines toto preventprevent theirtheir mutagenicmutagenic action ction [[14]. ] Copper Copper chlorophyllin chlorophyllin withwith hydrotalcitehydrotalcite [[34]34] andand graphenegraphene oxideoxide nanostructuresnanostructures [[35] ] exhibitsexhibits a a bactericidalb ctericidal effect.effect. SCCSCC adsorbed adsorbed ontoonto silksilk canc n serveserve asas natural n tural dye dye for for fibers fibers [1]. [ It ] is It also is worthalso worth mentioning mentioning that copolymer that copolymer of chlorophyllin of chlorophyllin sodium coppersodium salt, copper acrylic s acid,lt, acrylic n-butyl acid acrylate, n-butyl and acrylate, N-isopropylacrylamide and N-isopropylacrylamide is used as light is sensitive used as cation light exchangersensitive c for tion lysozyme exchanger purification for lysozyme [36]. purification [ ] DueDue toto their their hierarchical, hierarchical, anastomosing anastomosing structure, stru spongin-basedcture, spongin-b skeletons sed ofskeletons diverse spicule-freeof diverse marinespicule-free keratose marine sponges, keratose also sponges known also asbath known sponges,as bath represent sponges, promisingrepresent promising biological materialsbiological formaterials use in for several use in branches sever l branches of science, of scienc biomedicinee biomedicine and technology. and technology. These These demosponges demosponges are widelyare widely cultivated cultivated under under marine m ranching rine r nching conditions conditions and, consequently, and, consequently represent represent renewable renewable source ofsource special, of special, naturally naturally prestructured prestructured biological biological scaffolds. scaffolds. Nevertheless, Nevertheless to to date d te they they have h ve foundfound applicationapplication mostlymostly as s adsorbentsadsorbents [ [37,38], ], scaffoldsscaffolds forfor tissuetissue engineeringengineering andand regenerationregener tion [[39–42]39–42] andand templatestemplates for for development development of compositesof composites used inused electrochemistry in electrochemistry [43]. Therefore, [43]. Therefore it is necessary it is to further functionalize selected marine demosponge skeletons as special matrices in order to improve their surface properties and enable their use in various further applications. According to Int. J. Mol. Sci. 3 of necessaryInt. J. Mol. Sci. to2016 further, 17, 1564 functionalize selected marine demosponge skeletons as special m trices in order3 of 17 to improve their surface properties and enable their use in various further applications. According to our opinion, the combination of chlorophyllin with a support consisting of marinem rine demosponge spongin-based skeletonsskeletons makes makes it possibleit possible to obtain to obt a product in a product which combineswhich combines the desirable the propertiesdesir ble propertiesof both substrates: of both mechanical substrates: rigidity, mechanical high chemical rigidity and high thermal chemical resistance, nd biocompatibilitythermal resist nce and biocompatibilityantibacterial properties. and antib Our cterial study properties. focuses Our on obtainingstudy focuses novel, on functionalizedobt ining novel, dye-biopolymer functionalized dye-biopolymerhybrid material whichhybrid can material holds great which promise can holds for applications great promise in cosmetics, for applications medicine andin cosmetics, pharmacy medicine(as a drug and carrier pharm or wound cy (as dressing).a drug c rrier or wound dressing).

2. R Results sults and and Discussion Discussion

2.1. Adsorption Adsorption Tests Tests

2.1.1.Effect Effect of Contact Time Figure 2 shows the qu quantity ntity of dye (SCC) adsorbed on the surface of the selected fragments of H. communis marinemarine spongespongeskeletons skeletons as as a a function function of of time. time. The The adsorption adsorption process process was w carried s c rried out out for forchlorophyllin chlorophyllin solutions solutions at concentrations at concentrations of 100,of 200 and and 300 mg/L mg/L in in the the presence presence of of 0.1 M 1 M NaCl, NaCl in inaneutral a neutral environment, environment, for for a time a time of 1–90of min. min.

Figure 2 Effect of contact time and initial sodium copper chlorophyllin (SCC) concentration on the Figure 2. Effect of contact time and initial sodium copper chlorophyllin (SCC) concentration on the adsorption cap city of H. communis m rine sponge skeleton (0.1 M NaCl). adsorption capacity of H. communis marine sponge skeleton (0.1 M NaCl). The results show that in the initi l stage of the process there w s rapid rise in the quantity of dyes Theadsorbed results (q showt) for thate ch in concentration the initial stage an of ly the ed. process With anthere incre was se a rapidin the riseinitial in thedye quantity concentration, of dyes theadsorbed quantity (qt) adsorbed for each concentration lso increases analyzed. Reg rdless With of an the increase initial indye the concentration, initial dye concentration, the adsorption the efficiencyquantity adsorbed is close to also 100%. increases. This demons Regardlesstrates of the initialhigh sorption dye concentration, capacity of the H adsorptioncommunis skeletons efficiency withis close respect to 100%. to chlorophyllin. This demonstrates After the a highcertain sorption time (approximately capacity of H. communis 30 min ofskeletons the duration with respect of the experiment)to chlorophyllin. the value After stabilizes. a certain timeOur (approximatelyresults for the efficiency 30 min of of the adsorption duration of ofSCC the experiment) are better than the othersvalue stabilizes.previously Our reported results for for different the efficiency supports, of adsorption for example of SCC 5 are g/kg better SCC than on silk others fibers previously [ ] and reportedμmol/10 for mg different SCC on supports, chitosan for[33]. example, 49.75 g/kg SCC on silk fibers [1], and 5 µmol/10 mg SCC on chitosanIt was also [33]. investigated how chlorophyllin adsorption on H. communis sponge skeletons v ries as a functionIt was also of the investigated pH of the how solution chlorophyllin and of ionic adsorption strength. on TheH. communisresults aresponge presented skeletons in T ble varies 1. as a function of the pH of the solution and of ionic strength. The results are presented in Table 1. Int. J. Mol. Sci. 2016, 17, 1564 4 of 17

Table 1. Inﬂuence of pH and ionic strength on sodium copper chlorophyllin (SCC) adsorption capacity on H. communis spongin-based skeleton (contact time 60 min).

Dye Concentration Ionic Strength pH = 5 pH = 7 pH = 11 (mg/L) (mol/L NaCl) qt (mg/g) E (%) qt (mg/g) E (%) qt (mg/g) E (%) 25 6.25 100 5.58 89.24 0.25 3.95 50- 12.50 100 3.78 30.24 1.32 10.58 100 24.96 99.85 5.49 21.96 2.72 10.87 200 49.35 98.70 6.82 13.64 5.60 11.20 25 6.25 100 4.61 73.72 1.00 15.97 50 12.50 100 3.17 25.37 0.44 3.49 0.01 100 25.00 100 4.67 18.67 1.01 4.04 200 49.96 99.93 6.35 12.70 2.90 5.81 25 6.25 100 5.99 95.86 2.50 40.08 50 12.50 100 12.34 98.72 3.12 24.92 0.1 100 25.00 100 24.57 98.29 4.75 19.00 200 50.00 100 49.73 99.46 6.97 13.94

2.1.2. Effect of Initial Dye Concentration

The above results indicate noticeable variation in the values of qt and E. It was found that, independently of the other process parameters, the quantity of the dye adsorbed increases with increasing initial concentration of the dye solution. This pattern is related to the quantity of dye molecules adsorbed on the surface of the organic support. The higher the dye concentration, the larger the number of particles present in the solution that can become bound to the adsorbent. On the other hand, an increase in concentration leads to saturation of the active sites on the support, as a result of which a signiﬁcant quantity of dye particles are not adsorbed, causing a reduction in the process efﬁciency.

2.1.3. Effect of pH There are several factors that affect the stability of chlorophyll and chlorophyllin. As far as pH is concerned, all chlorophylls are most stable under alkaline conditions [44]. Analysis of the effect of pH showed that the type of environment plays a significant role in the process of adsorption of chlorophyllin on marine sponge skeletons studied. The quantity of dye adsorbed is greatest at pH = 5; for an initial concentration of 200 mg/L that value is 49.35 mg/g (without NaCl). By comparison, the qt values obtained in the same process conditions at pH = 7 (neutral pH) and pH = 11 are 6.82 and 5.60 mg/g, respectively. The process efficiency also indicates that an acidic environment is the best for adsorption (nevertheless, at pH = 3 chlorophylls will hydrolyze and lose color rapidly, and copper chlorophyllins will precipitate). In a neutral environment, there is large variation between the efficiency values obtained using solutions with concentrations ranging from 25 to 200 mg/L—the values for these extreme points are respectively 89.24% and 13.64%. For pH = 11, however, the values lie in the range of 3.95%–11.20%, while for pH = 5 they are close or equal to 100% (without NaCl). It can be seen that the decisive parameter is the acidity of the environment, not the initial dye concentration. The reason for this may be that in a low level pH environment the –NH2 groups of the proteinaceous + matrix undergoes protonation to –NH3 . In this case, it is possible for the cationic groups to be substituted by negatively charged ions of chlorophyllin. Thus, the adsorption takes place by way of electrostatic interactions.

2.1.4. Effect of Ionic Strength The effect of ionic strength is visible only when a larger quantity of NaCl (0.1 M) has been used. In an acidic or alkaline environment, there is a minimal increase in the quantity of dye adsorbed on the support as the ionic strength increases, since the decisive factor is the pH. However, a clear Int. J. Mol. Sci. 2016, 17, 1564 5 of 17 effect can be seen in a neutral environment. When the process is carried out with a dye solution with a concentration of 200 mg/L to which no NaCl has been added, the value of qt is 6.82 mg/g, but when 0.1 M NaCl is added the quantity of dye adsorbed rises to 49.73 mg/g. Similarly, an increase in the efﬁciency of the process can be observed. This behavior may be caused by several factors. According to the surface chemistry theory, repulsion between the adsorbed molecules and non-adsorbed molecules in the solution is opposite to the adsorption process, especially when the surface concentration is high. The presence of additional ions from salt in the medium decreases the repulsion between adjacent dye particles, allowing the adsorbed molecules on the surface to be closer to each other. In addition, the electric double layer, which surrounded both adsorbent and adsorbate, is compressed at high ionic strength, resulting in lowering or elimination of the repulsive energy barrier: thus, the van der Waals forces become signiﬁcant, leading to an increase in sorption of the dye on the particle surface [45,46]. This tendency is caused by a decrease in the repulsive electrostatic forces between the dye and the demosponge skeletons, causing an increase in adsorption when the ionic strength is increased [33].

2.2. Kinetic and Isothermal Studies To determine the kinetics of the adsorption process, pseudo-first order (PFO) and pseudo-second order (PSO) models were used. These two models basically include all steps in the process, such as external film diffusion, adsorption, and internal particle diffusion, so they are pseudo-models [47]. This analysis makes it possible to describe the dependence of the adsorption process on time. The linearized integral form of the pseudo-first order model is generally expressed as:

k log (q − q ) = log (q ) − 1 ·t (1) e t e 2.303 where qt and qe are the adsorption capacities at time t and at equilibrium respectively (mg/g), −1 k1 is the rate constant of pseudo-ﬁrst order adsorption (min ), and t is the contact time (min). − Plotting log (qe qt) versus t gives a linear relationship from which k1 and the predicted qe can be determined from the slope and intercept of the plot, respectively (Figure 3). The simpliﬁed and linearized form of the pseudo-second order model is:

t 1 1 · = 2 + t (2) qt k2qe qe · where k2 (g/(mg min)) is the second order rate constant of adsorption. The values of k2 and equilibrium adsorption capacity qe were calculated from the intercept and slope of the plot of t/qt versus t, according to Equation (2) and Figure 3 [48]. The kinetic process parameters are listed in Table 2.

Table 2. Kinetic constants of SCC adsorption on H. communis sponge skeletons at different dye concentrations (100, 200 and 300 mg/L; pH = 7; 0.1 M NaCl).

Initial Dye Concentration Kinetic Models Parameters 100 (mg/L) 200 (mg/L) 300 (mg/L)

qe,exp (mg/g) 24.84 49.73 74.57 q (mg/g) 0.17 14.62 12.80 Pseudo-First Order e,cal k1 (1/min) 0.006 0.084 0.076 r2 0.003 0.327 0.404

qe,cal (mg/g) 24.87 49.83 75.85 k (g/mg·min) 0.102 0.010 0.009 Pseudo-Second Order 2 r2 0.999 0.997 0.999 h 63.07 24.80 50.94 Int. J. Mol. Sci. 2016, 17, 1564 6 of 17 Int. J. Mol. Sci. 6 of

FigureFigure 3. Plots 3. Plots of theof the pseudo-ﬁrst pseudo-first order order (a () and) and pseudo-second pseudo-second orderorder ( b) models at at different different initial initial dyedye concentrations. concentrations.

The linear fit for t/qt vs. contact time and the r value for the pseudo-second order kinetic model The linear fit for t/q vs. contact time and the r2 value for the pseudo-second order kinetic model show that the dye dsorptiont kinetics can be pproximated as pseudo-second order Additionally show that the dye adsorption kinetics can be approximated as pseudo-second order. Additionally, the experimental values qe,exp fit the c lculated v lues qe,cal obtained from the linear plots of the thepseudo-second experimental valuesorder modelqe,exp betterfit the than calculated the pseudo-first values q e,calorderobtained values. In from the c the se linearof the adsorption plots of the pseudo-secondrate coefficient order determined model better using than the the PSO pseudo-first model, a order decrease values. can In be the observed case of the as adsorptionthe dye rateconcentration coefficient determined incre ses. This using is the due PSO to model, n incre a se decrease in the adsorbate’s can be observed compet asitiveness the dye for concentration the active increases.sites c pable This isof due adsorbing to an increase it. For the in coefficient the adsorbate’s(initi competitiveness l adsorption rate) for no the dependency active sites was capable found; of adsorbingthis results it. For from the the coefficient heterogeneityh (initial of the adsorption material used. rate) no dependency was found; this results from the heterogeneityThe most ofpopular the material isothermal used. theory for the adsorption of dyes onto biopolymers is the LangmuirThe most model popular [ isothermal ] The Freundlich theory forequ the tion adsorption is also frequently of dyes ontoapplied biopolymers in liquid–solid is the Langmuirsystems. modelAccording [49]. The to Freundlichthis theory, equationdye concentrations is also frequently on the adsorbent applied in will liquid–solid incre se so systems. long as According there is an to thisincre theory, se dyein the concentrations dye concentration on the in adsorbent the liquid will A basic increase assumption so long asof therethe L is ngmuir an increase theory in is the that dye concentrationsorption takes in the pl liquid. ce at specific A basic sites assumption within the of theadsorbent, Langmuir and theory therefore is that constitutes sorption monol takes place yer at specificdsorption. sites Adsorption within the equilibrium adsorbent, andis established therefore when constitutes an adsorbate-containing monolayer adsorption. phase h Adsorption s been in equilibriumcontact with is established the adsorbent when for an sufficient adsorbate-containing time, with the phaseadsorbate has concentration been in contact in withthe bulk the adsorbentsolution forin sufficient dyn mic time, equilibrium with the adsorbatewith the interface concentration concentration in the bulk [50]. solution Based inon dynamicexperimental equilibrium data, withadsorption the interface isotherms concentration were plotted [50]. Basedusing onthe experimentalFreundlich and data, Langmuir adsorption models. isotherms The correlation were plotted of the experimental dsorption data with number of adsorption models w s investigated to gain an using the Freundlich and Langmuir models. The correlation of the experimental adsorption data with understanding of the adsorption behavior a number of adsorption models was investigated to gain an understanding of the adsorption behavior. The nonlinear forms of the Freundlich (3) and L ngmuir (4) equations are presented below: The nonlinear forms of the Freundlich (3) and Langmuir (4) equations are presented below: (3) 1 · n qe = K f Ce (3) where Ce denotes the equilibrium concentration = of the∙ dye solution (mg/L) qe is the qu ntity of dye dsorbed at equilibrium (mg/g) nd Kf (mg/g) and n are the Freundlich constants. The values of Kf where Ce denotes the equilibrium concentration of the dye solution (mg/L), qe is the quantity of dye and n c n be determined from the intercept and gradient of the plot of log(qe) against log(Ce). adsorbed at equilibrium (mg/g), and Kf (mg/g) and n are the Freundlich constants. The values of Kf and n can be determined from the intercept and gradient of the plot of log(q ) against log(C ). e e (4) qm·b∙∙·Ce where Ce is the equilibrium concentration qofe =the= dye solution (mg/L), qm is the maximum adsorption(4) 1+∙+ b·Ce capacity, and b is the L ngmuir const nt (L/mg), which is c lculated from the intercept and wheredownwardCe is the linear equilibrium slope of concentration the graphs of C ofe/q thee and dye Ce solution (mg/L), qm is the maximum adsorption capacity,A and graphb is theof q Langmuire against constantCe for the (L/mg), adsorption which isotherms is calculated of chlorophyllin from the intercept on marine and downward sponge linearskeletons slope ofis theshown graphs in Figure of Ce /4.q e and Ce. A graph of qe against Ce for the adsorption isotherms of chlorophyllin on marine sponge skeletons is shown in Figure 4. Int. J. Mol. Sci. 2016, 17, 1564 7 of 17 Int. J. Mol. Sci. 7 of

Figure 4. Fit of experimental data to the L Langmuir ngmuir and Freundlich models.models

A v lue n or n implies that the dsorption process involves chemical or f vor ble A value n < 1 or n > 1 implies that the adsorption process involves a chemical or favorable physical physical process, respectively. The n v lue here is indicating a f vor ble process of adsorption process, respectively. The n value here is 2.27, indicating a favorable process of adsorption of SCC on of SCC on marine sponge skeletons. The Freundlich isotherm model fits the experimental dat marine sponge skeletons. The Freundlich isotherm model ﬁts the experimental data slightly better than slightly better than the Langmuir model (correl tion coefficient R 5, comp red with R the Langmuir model (correlation coefﬁcient R2 = 0.995, compared with R2 = 0.992 for the Langmuir for the Langmuir model). Maximum adsorption capacity, qm is relatively high nd equ l to model). Maximum adsorption capacity, qm, is relatively high and equal to 108.56 mg/g, whereas Kf, mg/g whereas Kf, which is a Freundlich constant related to adsorption cap city, is equal to which is a Freundlich constant related to adsorption capacity, is equal to 13.97 mg/g. The isothermal .97 mg/g. The isothermal parameters do not point clearly to either the Freundlich or the L ngmuir parameters do not point clearly to either the Freundlich or the Langmuir equation, which suggests equ tion, which suggests combined sorption mechanism. a combined sorption mechanism.

2.3.3. Desor Desorption tion Test First, adsorption of SCC onto H. communis sponge skeletons w was s c carried rried out (30 min; pH = 7; 0.11 M NaCl;N Cl four different concentrations:concentrations 25, 50, 100, 200 mg/L). mg/L) The adsorption efficiencyefﬁciency and nd dye concentration in the adsorbent phase were calculated.calculated. Next, desorption of the previously obtained samples was carried out. The process conditions were the same as for adsorption, except that water was used in place of the dye solution with 0.1 M NaCl. The process efficiency efﬁciency was was then then c calculated. lculated. The results indicate that the dye did not undergo desorption in the case of sampless mples with initialiniti l concentrations of 25,2 50, 100 and 200 00 mg/L. This This sugg suggestsests that that the the adsorption adsorption process is of a chemical nature, and nd that that the the chlorophyllin chlorophyllin was was deposited deposi permanentlyted permanently on the spongin-basedon the spongin-b sponge sed skeletons. sponge skeletons. 2.4. Structural Analysis 4. Structural Analysis The results of FTIR analysis of SCC, H. communis sponge skeletons and selected samples are presentedThe results in Figure of FTIR5. analysis of SCC, H communis sponge skeletons and selected samples are − presentedIn the in SCC Figure spectrum 5. (green line), absorption bands are observed in the range of 3600–2850 cm 1 attributableIn the SCC to stretching spectrum vibrations (green line) of O–H, bsorption N–H and bands C–H are bonds. observed The signalin the withrange maximum of 3 intensity cm− − attributableat 3405 cm 1tois assignedstretching to thevibrations stretching of vibrationsO–H, N H of OHand groupsC H bonds. in water The molecules, signal with while m the ximum bands − intensityat wavenumbers at 05 cm 2929− is and ssigned 2859 cm to the1 are stretching related tovibr symmetric tions of andOH groups asymmetric in w stretching ter molecules vibrations while − theof C–H bands bonds, at wavenumbers and that at 2 1632 cm nd 2851 stretching cm− are vibrations rel ted to ofsymmetric the carboxylate nd asymmetric anion. The stretching signals − vibrationsappearing inof theC range H bonds 1600–1300 and that cm at1 probably cm result− stretching from skeletal vibrations vibrations of the of carboxylate macrocyclic ringanion. of Thetetrapyrrole signals orappearing the alkyl substituentsin the range in the1 porphyrin 00 cm ring.− probably The chlorophyllin result from spectrum skeletcontains l vibrations a band of − macrocyclicat 1565 cm 1ring, which of tetrapyrrole can be attributedor the to lkyl C=C substituents and C=N bonds in the (skeletal porphyrin vibrations ring. The of thechlorophyllin porphyrin − spectrumring). The contains signals below a band 1000 t cm 51 cmgenerally− which arise can from be attributed molecular to motion C=C of nd carbon C=N bonds atoms, (skeletal and the − peaksvibrations at 991, of the 959 porphyrin and 924 cm ring).1 can The be signals attributed below to the1000 C–C cm− stretchinggener lly and arise bending from molecular vibrations motion of the − pyrroleof c rbon ring. atoms, The and peak the at 711peaks cm at1 results fromand vibrations 4 cm− can of be the attributed metal atom to the (copper) C–C stretching present in and the bendingstructure vibrations of the analyzed of the porphyrin.pyrrole ring. All The of peak the assignments t cm− results were made from in vibrations accordance of the with metal [21,51 atom,52]. (copper) present in the structure of the analy ed porphyrin. All of the assignments were made in accordance with [21,51,52]. Int. J. Mol. Sci. 2016, 17, 1564 8 of 17

Int. J. Mol. Sci. 8 of

Figure 5. FTIR spectr of SCC H. communis sponge skeletons and selected samples (sample Figure50 5. mg/L,FTIR sample spectra 2: of100 SCC, mg/L,H. sample communis 3: 200sponge mg/L; 60 skeletons min pH andneutral; selected 0 M samplesNaCl) (sample 1: 50 mg/L, sample 2: 100 mg/L, sample 3: 200 mg/L; 60 min; pH neutral; 0.1 M NaCl). On the FTIR spectrum for the spongin (black line) the wide band t 00 cm− is − Oncharacteristic the FTIR for spectrum deforming for stretching the spongin vibrations (black of the line), hydroxyl the widegroup. bandVibrations at 3500–3200 related to the cm 1 is presence of that group are lso responsible for the signals at wavenumbers and cm− . The characteristic for deforming stretching vibrations of the hydroxyl group. Vibrations related to the signal at cm− is ttributed to stretching vibrations of C–H bonds, nd that t cm− to −1 presencedeform of that tion vibrations group are of alsothe methyl responsible group –CH for the. The signals band t at wavenumbers 0 cm− indicates 1390the presence and 660 of cm . −1 −1 The signalstretching at 2930 vibrations cm is of attributed the carbonyl to stretchingchromophore vibrations C=O. The of presence C–H bonds, of aromatic and that rings at 1390 in the cm to −1 deformationstructure vibrations of the spongin of the is methylconfirmed group by the –CH two3 .b The nds band t at and 1640 1450 cm cm−indicates, both of which the presence are of stretchingattributed vibrations to vibrations of the carbonyl of coupled chromophore C C bonds C=O. in the The aromatic presence ring. of aromaticThe signal rings at in the cm structure− − of thecorresponds spongin is to confirmed stretching byvibrations the two of bandsC O bond at 1540s in carboxyl and 1450 groups. cm The1, both rem of ining which two arebands attributed at − − to vibrationsnd of10 coupled cm are C=C characteristic bonds for in stretching the aromatic vibrations ring. of The C–O signalbonds in at alcohols. 1230 cm 1 corresponds The FTIR spectrum of the H. communis sponge skeleton fragments studied with adsorbed to stretching vibrations of C–O bonds in carboxyl groups. The remaining two bands at 1070 and chlorophyllin−1 (cyan dark cyan and dark blue lines) contains signals origin ting both from the 1020 cmsupportare characteristicnd from the dye for stretchingThe wide bands vibrations in the of r C–O nge bonds in alcohols.cm− are characteristic for Thestretching FTIR vibr spectrum tions of ofOH the grouH.ps. communis The increasesponge in the intensity skeleton of the fragments signals at studiedwavenumbers with 2 adsorbed chlorophyllinnd 28 (cyan, cm− , reflecting dark cyan the andpresence dark of blue C–H lines) bonds, contains and of the signals signal originating at 1659 cm− both assigned from to the C=O support − and frombonds, the proves dye. the The effective wide adsorption bands in theof the range dye onto 3500–3200 the sponge cm skeleton.1 are characteristic There are also b for nds stretching at vibrations of OHnd groups. cm− Thewhich increase c n be attributed in the intensity to skeletal of vibrations the signals of the at porphyrin wavenumbers ring formed 2927 and − − − 2853 cmby C=C1, reflecting nd C=N bonds the presence (from SCC). of C–HThe peaks bonds, at and of nd the 35 signal cm indicate at 1659 stretching cm 1 assigned vibrations to C=O of C–O (their source is in marine sponge skeleton), while the signals with m xim at nd bonds, proves the effective adsorption of the dye onto the sponge skeleton. There are also bands at 01 cm− can be ssigned− to vibrations of C C bonds in the pyrrole ring The sign ls in the 1548, 1459 and 1403 cm 1, which can be attributed to skeletal vibrations of the porphyrin ring formed wavenumber range 700 50 cm− are generated by bonds formed between copper ions and −1 by C=Cchlorophyllin, and C=N bonds providing (from further SCC). confirm The peaks tion of atthe 1239 successful and 1035 immobili cm indicate tion of stretchingthe dye. vibrations of −1 C–O (theirItsource should isalso in be marine noted that sponge the small skeleton), shifts in while the m the ximum signals wavenumbers with maxima of certain at 916 peaks and (OH 801 cm can beand assigned NH groups, to vibrations C O bonds) of C–C m y bonds suggest in thethat pyrrolethe interactions ring. The between signals SCC in the and wavenumber H communis range − 700–550sponge cm skeletons1 are generated are generally by bonds based formedon ionic interactions. between copper This observation ions and chlorophyllin,is in agreement with providing furtherthe confirmation observations ofconcerning the successful changes immobilization in process efficiency of the dye.depending on changes in the pH of Itthe should solution. also be noted that the small shifts in the maximum wavenumbers of certain peaks The results of energy-dispersive X-r y spectroscopy (EDS) are given in T ble (OH and NH groups, C=O bonds) may suggest that the interactions between SCC and H. communis The marine sponge skeleton studied here is composed mainly of c rbon, nitrogen and oxygen. spongeThese skeletons elements are make generally up protein based structure on ionic (spongin interactions.), which This is the observation main component is in agreementof the skeleton with the observationsof H. communis concerning. EDS changesanalysis in lso process reve ls efficiencythe presence depending of chlorine on changes luminum, in silicon, the pH sulfur of the and solution. Theiodine results (in the of energy-dispersiveform of 3,5-diiodotyrosine, X-ray spectroscopyc lled iodogorgonic (EDS) acid) are given[53]. It in h Table s been3 .proved that Thehalogens marine exist sponge in combination skeleton with studied org nic here comp is composedonents in demosponges mainly of carbon, [54]. The nitrogen data from and EDS oxygen. Theseanalysis elements confirm make indirectly up a protein the structureeffectiveness (spongin), of adsorption which process, is the main elements component both from of SCC theskeleton and of H. communisH. communis. EDS skeleton analysis are also presented reveals in the obtained presence hybrid of material. chlorine, aluminum, silicon, sulfur and iodine (in the form of 3,5-diiodotyrosine, called iodogorgonic acid) [53]. It has been proved that halogens exist in combination with organic components in demosponges [54]. The data from EDS analysis confirm indirectly the effectiveness of adsorption process, elements both from SCC and H. communis skeleton are presented in obtained hybrid material. Int.Int. J. Mol. J. Mol. Sci. Sci.2016 , 17, 1564 9 of9 of 17

Table 3. EDS nalysis results Table 3. EDS analysis results. Cont nt of Elements by ight (%) Element Sponge Skeleton SS + SCC SS + SCC Chlorophyllin Content of Elements by Weight (%) Element Sponge(SS) Skeleton (SS mg/L) + SCC (SS 0 mg/L) + SCC Chlorophyllin C 8(SS) (400 mg/L) (1000 mg/L) NC 4. 84.66 81.67 88.13 82.85 ON 4. 4.48 3.18 0.20 2.56 CuO 4.84 10.37 - 2.62 8.08 Cu 2.80 - 0.26 0.53 ClCl 0.42 0.34 5.45 1.52 NaNa 2.79 - - 1.91 0.80 AlAl - - 1.17 0.27 0.77 Si - 0.25 0.04 0.10 Si - S - 1.15 0.31 0.87 S I- - 1.87 0.82 1.92 TotalI - 100.00 100.00 100.00 100.00 Tot l The 13C CP/MAS NMR spectra of H. communis sponge skeletons and selected hybrid materials H. communis (obtainedThe fromC CP/MAS 750 mg/L NMR SCC, spectra which of give 101.5 mgsponge of dye skeletons on 1 gand of H.selected communis hybridskeleton) m terials are (obtained from 750 mg/L SCC, which give 101 mg of dye on g of H communis skeleton) re presented in Figure 6. The additional signals (in comparison with (a), written horizontally) visible presented in Figure 6. The additional signals (in comparison with ( ), written horizontally) visible on on spectrum (b) come from the SCC adsorbed on the sponge skeletons studied. Based on previously spectrum (b) come from the SCC adsorbed on the sponge skeletons studied. Based on previously published data [55,56] they can be assigned to: C1, C6 or C16 carbons from the porphyrin macrocycle published dat [55 ] they can be assigned to: C , C6 or C16 c rbons from the′ porphyrin m crocycle (154.8 ppm); C13 (126.7 ppm) carbon chains with double bond; C17 and C17 from the reduced phytol ( ppm) C13 ′(1 .7′′ ppm) carbon chains′ with′ double′ bond; C1 nd C17′ from the reduced chainphytol (36.3 chain ppm); (3 C8 3 ,ppm); C8 (15.2; C ′, 14.3C ′′( ppm), C2 .3 ,ppm) C7 , C12 C ′, (9.5C ′, ppm)C12′ (9. from ppm) the macrocycle’s from the macrocycle’s short carbon chainshort substituent. carbon chain Moreover, substituent. adsorption Moreover, of adsorpti the dyeon induces of the dye slight induces differences slight indifferences the values in ofthe the signalsvalues which of the are signals present which in the are spectra present of in both the thespectra investigated of both the marine investigated sponge skeletonsmarine sponge and the hybridskeletons material and the developed. hybrid m terial developed.

Figure 6. C CP/MAS NMR spectra of H. communis sponge skeletons ( ) and developed hybrid Figure 6. 13C CP/MAS NMR spectra of H. communis sponge skeletons (a) and developed hybrid material (b) (75 mg/L; 30 min; pH neutral; 0 1 M NaCl) (additional signals are written horizontally). material (b) (750 mg/L; 30 min; pH neutral; 0.1 M NaCl) (additional signals are written horizontally). Data from spectroscopic analysis provides important information both about H. communis structureData from and possible spectroscopic mechanism analysis of interaction provides be importanttween SCC informationand spongin-based both aboutspongeH. skeletons. communis structureHydrogen and bonds possible formation mechanism and electrost of interaction tic inter ction between play SCC key role and in spongin-basedthis process Indirect sponge skeletons.confirm tionHydrogen of this assumptions bonds formation is lso andprovided electrostatic by results interaction from adsorption play a studies key role in this process. Indirect conﬁrmation of this assumptions is also provided by results from adsorption studies. Int. J. Mol. Sci. 2016, 17, 1564 10 of 17 Int. J. Mol. Sci. 10 of

FigureFigure7 presents presents the the illustrative illustrative combinationcombination of typic typical l amino amino acids, acids, which which build build the the sponging sponging structurestructureInt. J. Mol. as as Sci. well well as as possible possible interactioninter ction betweenbetween the functional functional group group of of these these amino amino acid10 acid of and and SCCSCC molecule. molecule. Figure presents the illustrative combination of typic l amino acids, which build the sponging structure as well as possible inter ction between the functional group of these amino acid and SCC molecule.

Figure 7. Proposed mechanism of interaction between SCC and spongin-based sponge skeletons Figure 7. Proposed mechanism of interaction between SCC and spongin-based sponge skeletons (typical spongin-build amino acids are marked in frames). (typicalFigure spongin-build 7. Proposed amino mechanism acids of are interaction marked in between frames). SCC and spongin-based sponge skeletons (typical spongin-build amino acids are marked in frames). Sodium copper chlorophyllins are rel tively therm lly stable [ ] As shown in Figure 8 the thermogramSodium copper of SCC chlorophyllins contains three discrete are relatively char cte thermallyristic decomposition stable [51]. steps As shown The first in Figureweight 8loss,a, the Sodium copper chlorophyllins are rel tively therm lly stable [ ] As shown in Figure 8 the thermogramof bout % of between SCC contains 85 and three 115 °C discrete can simply characteristic be ascribed decomposition to the escape steps.of moisture The ﬁrst or air weight adsorbed loss, of thermogram of SCC contains◦ three discrete char cteristic decomposition steps The first weight loss, aboutin ofthe 4%, bouts between mple % between[ 7]85 and According 85 115 andC 115 canto °C [51] simply can thesimplybe second ascribed be as decompositioncribed to the to escapethe escape step, of moistureof inmoisture the ortemperature or air air adsorbed adsorbed range in the ◦ samplein [the57 ].°C,s According mple with [a 7]percentage According to [51], the loss to second [51]of the %, decomposition msecond y be decomposition ttributed step, to in thestep, elimin temperature in the tion temperature of range Cu ions.240–370 range The C, withmaximum a percentage rate°C, with of loss m a sspercentage of loss 20%, ( bout may loss beof %) attributed of %, SCCm y occurredbe to ttributedthe eliminationat to the elimin °C of Cu nd tion2+ matchesions. of Cu Thean ions. observed maximum The ◦ rateexothermic ofmaximum mass losspeak rate (about (Figureof m ss40%) 8b)loss ofwhich( boutSCC is occurred %)indicative of SCC at occurred 390–510of the rupture atC and and matches °Cdegradation nd anmatches observed of thean observedporphyrin exothermic peakmexothermic (Figurecrocycle8 [58].b), peak which From (Figure is a temperature indicative 8b) which of ofis the indicative rupture °C there of and the is degradation anotherrupture smalland ofdegradation drop the porphyrin in m of ss the(to macrocycle porphyrin %) which [58 ]. ◦ Frommaym a temperaturebe crocycle ssoci [58]. ted ofFrom with 700 a combustiontemperatureC, there is anotherofof the °Corga small therenic drop ismatrix. another in mass Nevertheless small (to drop 60%), in whichmSCC, ss (to maylike %)the be which associatedsponge withskeletons, combustionmay be does ssoci ofnot ted the show with organic completecombustion matrix. loss Nevertheless,of ofthe ma orgass nicin SCC,TG-DTAmatrix. like Nevertheless measurements, the sponge SCC, skeletons, because like the does of sponge the not coal show formationskeletons, [ does ] not show complete loss of mass in TG-DTA measurements, because of the coal complete loss of mass in TG-DTA measurements, because of the coal formation [58]. formation [ ]

FigureFigure 8. TG8. TG ( )( and) and DTA DTA ( b()b )curves curves for for H.H. communiscommunis sponge skeletons,skeletons, SCC, SCC, and and selected selected samples samples Figure 8. TG (a) and DTA (b) curves for H. communis sponge skeletons, SCC, and selected samples (sample(sample a: 400a: 400 mg/L, mg/L, sampleb sampleb mg/L; mg/L; 30 30 min; min; pHpH neutral; 00 1 1 MM NaCl) NaCl) (sample a: 400 mg/L, sampleb: 750 mg/L; 30 min; pH neutral; 0.1 M NaCl). Int. J. Mol. Sci. 2016, 17, 1564 11 of 17

Int.The J. Mol. thermogram Sci. of the H. communis sponge skeletons have been recently described in detail11 of in [38]. Int. J. Mol. Sci. 11 of The thermogram of the spongin-based skeleton studied in this work with adsorbed chlorophyllin The thermogram of the H. communis sponge skeletons have been recently described in det il in [38]. shows threeThe characteristicthermogram of decompositionthe H. communis stages.sponge skeletons The first have mass been loss, rece ofntly around described 10%, observedin det il in between[38]. The thermogram◦ of the spongin-b sed skeleton studied in this work with dsorbed chlorophyllin 80 andThe 250thermogramC, is associated of the spongin-b with evaporation sed skeleton of thestud residualied in this water work in with the sample. dsorbed Thechlorophyllin second stage shows three characteristic decomposition◦ stages The first mass loss, of around 10%, observed shows three characteristic decomposition stages The first mass loss, of around 10%, observed occursbetween in the 80 temperature and 50 °C, rangeis associ 300–600 ted withC, ev with por aation 40% of mass the loss,residual which water may in bethe attributedsample. The to the between 80 and2+ 50 °C, is associ ted with ev poration of the residual water in the sample. The eliminationsecond stage of Cu occursions in or the to temperature thermal decomposition range 00 of °C, the with organic phase. % m ss Another loss, which small m mass y be loss second stage occurs in the temperature range◦ 00 °C, with % m ss loss, which m y be (of aroundattributed 10%) to the occurs elimination in the rangeof Cu 800–1000ions or to thermC, this l decompositio may reflect combustionn of the organic of thephase. organic Another matrix. attributed to the elimination of Cu ions or to therm l decomposition of the organic phase. Another Thesmall thermogram m ss loss shows (of around that when 10%) theoccurs dye in is the applied r nge to80 the H. communis °C, this msponge y reflect skeletons combustion their of thermalthe small m ss loss (of around 10%) occurs in the r nge 80 °C, this m y reflect combustion of the stabilityorganic increases, matrix. andThe thethermogram greater the shows quantity that ofwhen adsorbed the dye chlorophyllin, is applied to the greaterH. communis the resistance sponge to organic matrix. The thermogram shows that when the dye is applied to the H. communis sponge thermalskeletons decomposition. their thermal st bility incre ses, and the greater the quantity of adsorbed chlorophyllin, skeletons their thermal st bility incre ses, and the greater the quantity of adsorbed chlorophyllin, theUsing greater an opticalthe resistance microscope to thermal and decomposition. a scanning electron microscope (SEM), photographs were taken the greater the resistance to thermal decomposition. Using an optical microscope and a scanning electron microscope (SEM), photographs were to enableUsing precise an analysisoptical microscope of the morphology and a scanning and microstructure electron microscope of the (SEM sponge), photographs skeleton before were and t ken to enable precise analysis of the morphology nd microstructure of the sponge skeleton before aftert the ken adsorption to enable precise process. analysis of the morphology nd microstructure of the sponge skeleton before and after the adsorption process. andThe after photographs the adsorption of the process. spongin-based skeleton fragments (Figures 9a and 10a,b) indicate their The photographs of the spongin-b sed skeleton fragments (Figure 9 and Figure a,b) indicate typical fibrous,The photographs reticulate of structure. the spongin-b Single sed fibers, skeleton composed fragments of (Figure microfibers, 9 and combineFigure a,b) into indicate a complex their typical fibrous, reticulate structure. Single fibers, composed of microfibers, combine into a hierarchicaltheir typical network. fibrous, Subsequent reticulate structure. photographs Single(Figure fibers, composed10c,d) show of microfibers, that the fibers combine are covered into a by complex hierarchic l network. Subsequent photographs (Figure 10c,d) show that the fibers are complex hierarchic l network. Subsequent photographs (Figure 10c,d) show that the fibers are a layercovered of the by adsorbed layer of dyethe adsorbed (750 mg/L, dye 30(750 min, mg/L pH neutral, min, pH 0.1 neutral M NaCl). 1 M N Cl). covered by layer of the adsorbed dye (750 mg/L min, pH neutral 1 M N Cl).

Figure 9. Light microscopy images of H. communis sponge skeletons before ( ) and after (b) FigureFigure 9. Light 9. Light microscopy microscopy images images of H. of communis H. communisspongesponge skeletons skeletons before before (a) and ( after) and (b after) adsorption (b) adsorption of SCC. of SCC.adsorption of SCC.

Figure 10. SEM images of purified H. communis sponge skeletons before ( b) and after (c d) Figure 10. SEM images of purified H. communis sponge skeletons before ( b) and after (c d) Figureadsorption 10. SEM of images SCC, at of different puriﬁed magnifications.H. communis sponge skeletons before (a,b) and after (c,d) adsorption of SCC,adsorption at different of SCC, magniﬁcations. at different magnifications. Int. J. Mol. Sci. 2016, 17, 1564 12 of 17

2.5. Antibacterial Tests The results of antibacterial activity for the analyzed materials against Staphylococcus aureus are presented and described below. Tetracycline antibiotic was used as a positive control. The results demonstrate that the H. communis sponge skeleton–SCC hybrid material reduced the growth of the above-mentioned gram-positive bacteria. Although the effect is not observed for the first two samples (0.1 and 1 mg), antibacterial activity emerged and increased with higher contents of SCC (for 5 and 10 mg of weighted portion of hybrid material the inhibition zone diameter is equal to 13 and 14 mm, respectively). The absence of antibacterial activity does not imply the absence of bioactive components, but active constituents may be present in insufficient quantities to inhibit cell growth. Lack of activity can thus only be proven by using large doses [59]. According to [60], the diameter of the halo zone indicates that the hybrid material exhibits good antibacterial activity. Also, an extract from this sponge species does not exhibit antibacterial properties against Straphylococcus aureus, as reported in [61]. In comparison, 30 µg of Tetracycline reduce the growth of S. aureus less that examined hybrid material, the halo zone is 7 mm. H. communis sponge skeletons without any modification showed no antibacterial activity against the tested bacterial species. The hybrid of SCC and H. communis sponge skeleton is not so active as SCC (as results from conversion of the actual quantities of SCC in 100 µL of solution and 100 µL of suspension), inhibition zone diameter is equal to 12 and 14 mm, for 100 mL of 100 and 400 mg/L of sodium copper chlorophyllin solution, respectively. Nevertheless, there are other factors which make this material much more useful than the pure dye, for example, its insolubility provides the possibility of reusing it. The same behavior was also reported previously [35]. The antibacterial activity of SCC can be explained by its excellent photosensitizing properties. When the molecules are activated by visible light, they generate singlet oxygen, which is cytotoxic to most living cells. When these short-life free radicals are close enough to the cell surface, they can trigger extensive disruption in the cell membrane, resulting in cell death [5]. Moreover, similar experiments were performed against gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, using both SCC alone and obtained hybrid material, but any antimicrobial activity was observed (data not provided). Different activity of SCC against gram-positive and gram-negative bacteria could be explained by different cell membrane structures. In both cases, the cell wall is constructed from the polymer peptidoglycan, which is thicker in gram-positive bacteria (S. aureus). In contrast, gram-negative bacteria (E. coli and P. aeruginosa) have a thin layer of peptidoglycan, but it is located between the cytoplasmic membrane and an extra, second membrane called the outer membrane made of phospholipids and lipopolysaccharides [62,63], which probably made them resistant to SCC activity.

3. Materials and Methods

3.1. Materials Marine demosponges of the species Hippospongia communis, purchased from INTIB GmbH (Freiberg, Germany), were originally collected in the Mediterranean Sea (Tunisia). To ensure the uniformity, all the samples were prepared according to the same procedure. The sponge material was washed with distilled water to remove salts. After that, it was stored for 3 days in 3 M HCl at room temperature with the aim to dissolve possible calcium carbonate-containing contaminations. ◦ The material was then rinsed with distilled water up to pH 6.5 and dried during 24 h at 50 C. Chlorophyllin sodium copper (SCC) salt was from Sigma Chemical Co. (St. Louis, MO, USA) and used as supplied. The stock solution was prepared by dissolving an appropriate amount of dye in 1 L of distilled water. Experimental solutions of desired concentrations were obtained by successive dilutions with distilled water. Other chemicals were of reagent grade and used without further puriﬁcation. Int. J. Mol. Sci. 2016, 17, 1564 13 of 17

3.2. Adsorption and Desorption Tests Batch experiments were performed to investigate the effect of contact time and to determine kinetic parameters. Adsorption experiments were performed using glass bottles containing appropriate quantities of marine sponge skeleton and dye solution. The initial concentration of the dye was 100, 200 and 300 mg/L. SCC was dissolved in water. After different time intervals, the samples were filtered off under vacuum and analyzed using a UV-VIS spectrophotometer (Jasco V750, Jasco, Tokyo, Japan) at maximum absorbance wavelength 405 nm. After filtration each sample was left to dry in room temperature for 48 h. Dye concentration in the adsorbent phase at a specific time (qt) (5) and the adsorption efficiency (E) (6) were calculated as:

(C − C ) ·V q = 0 t (5) t m C − C E (%) = 0 t ·100% (6) C0 where C0 and Ct are the concentrations of the dye in the solution before and after sorption, respectively (mg/L), V denotes the volume of the solution (L), and m is the mass of sorbent (g). The effect of pH on the adsorption of SCC from aqueous solution by selected fragments of the marine sponge skeleton was similarly investigated. The pH was adjusted to 3, 5, 7, 9 and 11 using either 0.1 M HCl or 0.1 M NaOH. The effect of ionic strength on dye sorption was studied by adding NaCl (0.01 and 0.1 M) to 50 mL of SCC solution. Adsorption isotherms were obtained by placing the samples of selected sponge skeletons in a series of flasks containing 50 mL of dye solution at different initial concentrations (25–1000 mg/L + 0.1 M NaCl) prepared from stock solutions. After shaking (30 min), the residual concentration of the dye was estimated. The next step was the evaluation of the hybrid material’s stability (desorption tests). To a conical flask containing 50 mL water, a portion of the obtained hybrid material was added. The suspension was shaken for 4 h at room temperature. The suspension was filtered off under reduced pressure. In the filtrate, the concentration of the soluble components was evaluated by absorbance measurements, and the concentration of the eluted dye was determined from the calibration curve.

3.3. Analysis The dye-biopolymer hybrid material and precursors (dye and marine sponge skeleton) were also subjected to FTIR spectral analysis (using a Vertex 70 spectrometer, Bruker, Billerica, MA, USA). The materials were analyzed in the form of tablets, made by pressing a mixture of 250 mg anhydrous KBr and 1 mg of the analyzed substance under a pressure of approximately 10 MPa. The investigation − − was performed over a wavenumber range of 4000–400 cm 1 (at a resolution of 0.5 cm 1). The surface composition was analyzed using a Tescan scanning electron microscope equipped with a PTG Prism Si(Li) (Princeton Gamma Tech., Princeton, NJ, USA) energy-dispersive X-ray spectrometer (EDS). Before the analysis, samples were placed on the ground with a carbon paste or tape. The presence of carbon materials is needed to create a conductive layer which ensures the delivery of electric charge from the sample. Cross Polarization Magic Angle Spinning Nuclear Magnetic Resonance (13C CP/MAS NMR) analysis was performed using a DSX spectrometer (Bruker, Billerica, MA, USA). For this purpose 100 mg of a sample was located in a rotator, made of ZrO2, 4 mm in diameter and centrifuged at a spinning frequency of 8 kHz. 13C CP/MAS NMR spectra were measured at 100.63 MHz in a standard 4 mm MAS probe using a single pulse excitation with high power proton decoupling (pulse repetition 10 s, spinning speed 8 kHz). Int. J. Mol. Sci. 2016, 17, 1564 14 of 17

The heat effects were determined by thermogravimetric analysis (TG and DTA) on a thermoanalyzer ◦ (Jupiter STA 449F3, Netzsch, Selb, Germany). Samples (approximately 5 mg) were heated to 1000 C ◦ from room temperature at a rate of 10 C/min, in a nitrogen atmosphere. Images were made by means of a Keyence VHX-5000 (Keyenece, Osaka, Japan) digital optical microscope. The microstructures were studied under a Tescan Vega 5135 scanning electron microscope, in order to obtain data on surface morphology and structure.

3.4. Antibacterial Tests Tests were carried out on systems composed of a dye and H. communis sponge skeletons (obtained from a 1000 mg/L SCC solution). Weighed portions were suspended in 1 mL of sterile water. The control consisted of a tetracycline antibiotic (30 µg), SCC solutions (100 and 400 mg/L) and sponges without the addition of dyes. Suspensions of indicator microorganisms, secured and stored at ◦ −20 C, were thawed at room temperature, and then transferred to test tubes containing 10 mL of stock ◦ medium with the addition of 2% glucose (Oxoid, Basingstoke, UK). Culture took place at 35 ± 2 C for 24 h. The tested bacterial suspensions containing 106 cfu/mL were spread on Mueller-Hinton Agar (Oxoid) and left for 15 min to absorb the microorganisms on the surfaces. The indicator microorganisms used in the test was Staphylococcus aureus species. At the next stage of testing, on the surface of the prepared medium, wells 9 mm in diameter were made, into which 100 µL of suspension (in the case of the weighed samples) or 100 µL of solution (in the case of SCC) was applied. The Petri dishes with ◦ the applied samples were then incubated at a temperature of 35 ± 2 C for 24 h. The antibacterial activity was evaluated based on the diameter of the zone in which growth of the indicator bacteria was inhibited.

4. Conclusions Marine sponge skeletons isolated from Hippospongia communis are suitable as a solid support for sodium copper chlorophyllin. SCC can be tightly fixed on the support surface. The results of analysis suggest electrostatic interaction and hydrogen bonding between these two constituents. Adsorption conditions, especially pH and addition of salt, have a considerable effect on the results obtained. The adsorption rate was very high in the initial period of contact, due to the availability of reactive sites on the adsorbent. After equilibrium, no significant changes in adsorption efficiency are observed, because of saturation of the adsorbent after a longer contact time. The kinetics of the adsorption process is best described by a pseudo-second order model. Isothermal parameters do not indicate clearly either a Freundlich or a Langmuir model, which suggests a complex sorption mechanism. The results of TG, 13C CP/MAS NMR and FTIR analysis indirectly confirm the effectiveness of the process, as does EDS analysis, which also supplies additional information concerning the structure of the marine sponges. The antibacterial activity of the hybrid made of SCC with marine demosponge skeleton—an environmentally friendly and cost-effective material—against S. aureus was investigated and proved for the first time.

Acknowledgments: This work was partially financially supported by Poznan University of Technology research grant no. 03/32/DS-MK/0610, and the DFG Grant EH 394-2, Germany. Authors are grateful to Vasilii Bazhenov and the Keyence Co. for the optical microscopy investigations. Author Contributions: Małgorzata Norman conceived and designed the experiments, developed the results and wrote the manuscript; Przemysław Bartczak kinetic analysis; Jakub Zdarta FTIR analysis; Wiktor Tomala and Barbara Zurańskaperformed˙ adsorption experiments; Anna Dobrowolska and Katarzyna Czaczyk antibacterial tests; Adam Piasecki EDS analysis; Hermann Ehrlich and Teofil Jesionowski planning and coordination of this research. Conflicts of Interest: The authors declare no conflict of interest. Int. J. Mol. Sci. 2016, 17, 1564 15 of 17

References

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© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). Open Chem., 2016; 14: 243–254

Research Article Open Access Małgorzata Norman, Jakub Zdarta, Przemysław Bartczak, Adam Piasecki, Iaroslav Petrenko, Hermann Ehrlich, Teofil Jesionowski* Marine sponge skeleton photosensitized by copper phthalocyanine: A catalyst for Rhodamine B degradation

DOI 10.1515/chem-2016-0025 received October 2, 2016; accepted November 4, 2016. 1 Introduction

Abstract: We present a combined approach to photo- Phthalocyanines are macrocyclic, aromatic compounds assisted degradation processes, in which a catalyst, based on the porphyrin structure, in which four indole

H2O2 and UV irradiation are used together to enhance rings are connected by azomethine bridges. Because of the oxidation of Rhodamine B (RB). The heterogeneous their specific chemical structure, they exhibit unique photocatalyst was made by the process of adsorption properties, which make them suitable for a wide range of of copper phthalocyanine tetrasulfonic acid (CuPC) applications. Metalphthalocyanines possess high chemical onto purified spongin-based Hippospongia communis and thermal stability, high reactivity, redox properties and marine sponge skeleton (HcS). The product obtained, high molar absorption coefficients [1-4]. Moreover, the CuPC-HcS, was investigated by a variety of spectroscopic type, position, and number of substituents and the central (carbon-13 nuclear magnetic resonance 13C NMR, Fourier atom can change the physical and chemical properties of transform infrared spectroscopy FTIR, energy-dispersive a phthalocyanine molecule [5]. X-ray spectroscopy EDS) and microscopic techniques Metalphthalocyanines are used as sensors [2,5], (scanning electron microscopy SEM, fluorescent and photosensitizers in photodynamic therapy and solar cells optical microscopy), as well as thermal analysis. The (DSSC) [3] and dyes (photochromic substances, fluorescent study confirms the stable combination of the adsorbent probes in sensing or in vitro imaging applications). They and adsorbate. For a 10 mg/L RB solution, the percentage are semiconductive and light-absorbing electron donor degradation reached 95% using CuPC-HcS as a compounds, and can serve in electronic and optoelectronic heterocatalyst. The mechanism of RB removal involves devices [6], photovoltaic cells, electrode modifications adsorption and photodegradation simultaneously. [7,8], liquid crystals [9] and data storage systems [10,11]. Phthalocyanines are also commonly used as catalysts, in Keywords: Hippospongia communis, spongin, organic synthesis [12], degradation of organic pollutants metalphthalocyanine, photocatalysis, Rhodamine B (e.g. 2,4-D acid [13], synthetic dyes [14,15]), and oxidation of harmful and undesirable compounds (e.g. bisphenol A [16], phenols [17], thiols [18,19]). Metalphthalocyanines exhibit high catalytic activity *Corresponding author: Teofil Jesionowski: Poznan University of Technology, Faculty of Chemical Technology, Institute of Chemical (in a so-called biomimetic catalytic system) even in Technology and Engineering, Berdychowo 4, 60965 Poznan, Poland, ambient conditions [20]; in addition they are relatively E-mail: teoﬁ[email protected] easy to synthesize and commercially available [21]. Małgorzata Norman, Jakub Zdarta, Przemysław Bartczak: Poznan However, one limitation of these compounds is that University of Technology, Faculty of Chemical Technology, Institute they are inconvenient to use, since they are generally of Chemical Technology and Engineering, Berdychowo 4, 60965 available only as powder, in solution (which hinders their Poznan, Poland Adam Piasecki: Poznan University of Technology, Faculty of separation from the solution and catalyst recycling) or in Mechanical Engineering and Management, Institute of Materials the form of thin film [22-24]. Moreover, some molecules are Science and Engineering, Jana Pawła II 24, 60965 Poznan, Poland prone to aggregation, which greatly decreases the number Iaroslav Petrenko, Hermann Ehrlich: TU Bergakademie Freiberg, of active sites for catalysis [25]. A better strategy for their Institute of Experimental Physics, Leipziger 23, 09599 Freiberg, practical use might be to attach them to a suitable support Germany

© 2016 Małgorzata Norman et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivsAuthenticated | [email protected] License. author's copy Download Date | 12/9/16 8:09 AM 244 Małgorzata Norman et al. material. Adsorption of metalphthalocyanines on a solid RB using marine sponge skeleton has not previously been support is an effective way to remedy the drawbacks studied. of the homogeneous catalyst and enable the creation of a heterogeneous system (possibility of reutilization, increase in the surface area of the catalyst). Adsorbents 2 Experimental used to date include zeolites [26], silica [13], TiO2 [27], carbon materials [19,28], polymers [29] and fabrics [1]. In the present work we decided to use 3-dimensional 2.1 Materials fibrous proteinaceous skeletons (built from a specific protein – spongin) of marine demosponge origin. In Specimens of Hippospongia communis demosponge were comparison with traditional supports, this material collected on the Mediterranean coast in Tunisia and does not have to be synthesized, and the process supplied by INTIB GmbH (Germany). Pieces of selected of adsorption is fast, facile and takes place in mild sponges were prepared by washing with sea water, conditions. Mediterranean Hippospongia communis followed by drying and rinsing again with fresh water sponge possess a fibrous, anastomosing, spatial, reticular five times. To remove contaminants (such as CaCO3) the structure. The structure of spongin is multileveled - pieces were immersed in a 3M HCl solution for 72 h at single fibers, composed of microfibers, combine into a room temperature and finally washed with distillated complex hierarchical network. Spongin fibers may range water up to a neutral level of pH. Copper phthalocyanine- in thickness from a few mm to about 10-15 mm. The 3,4′,4″,4″′-tetrasulfonic acid tetrasodium salt (CuPC) and combination of this renewable material with a relative Rhodamine B (RB) were purchased from Sigma Aldrich large internal surface area (between 25 and 34 m2 for a 3- to (Germany). Other chemicals were of reagent grade and 4-gram skeleton [31]) with copper phthalocyanine enable used as supplied. the production of the heterocatalyst. Sponges (phylum Porifera) are one of the phylogenetically oldest multicellular organisms, 2.2 Adsorption and desorption process which evolution dates back to 600 million years ago. They are aquatic animals, currently described in four To evaluate the adsorption properties of the purified classes: Demospongiae, Calcarea, Hexactinellida and marine sponge skeleton with respect to CuPC, a series of Homoscleromorpha [30]. Demosponges skeletons are adsorption experiments were performed. Typically, sponge fibrous and contain species dependent chitin, or spongin as pieces of 0.1 g were shaken with 25 mL of experimental main organic constituents. Spongin contains both collagen- solutions of desired concentrations (for kinetic studies and keratin-like structural proteins that are responsible for 50, 100 and 200 mg/L) at pH=2. After predetermined time the rigidity of the sponge skeleton [31,32]. Because of their intervals, the adsorbed quantity (mg/g) of CuPC at time t, unique spatial structure and properties (e.g. high thermal qt, was calculated from the following equations: stability) spongin-based skeletons of diverse demosponges are currently the subjects of numerous studies related to (1) tissue engineering [33,34], as well as to Extreme Biomimetics [35,36]. Due to diverse marine ranching techniques used for (2) the cultivation of spongin-based demosponges, spongin is the renewable source (which affect their relatively low where C0 and Ct are the concentrations of the dye in the price) of proteinaceous scaffolds with good perspective for solution before and after sorption (mg/L), V is the volume practical applications. of the solution (L), and m is the mass of sorbent (g). In the present work, for the first time, copper Initial dye concentrations for kinetic studies ranged from phthalocyanine tetrasulfonic salt (CuPC) was successfully 50 to 200 mg/L (pH=2). adsorbed onto purified marine bath sponge skeleton In order to study the adsorption isotherm, 0.1 g of from the species Hippospongia communis. In addition, marine sponge skeleton was kept in contact with 25 mL we investigated the photocatalytic properties of the of dye solution at different concentrations (50, 100, 200, resulting systems for organic pollution degradation, using 300, 400 and 500 mg/L) at pH=2 for 240 min with constant the synthetic dye Rhodamine B as the target compound. shaking at ambient temperature. Although UV light-assisted photocatalytic degradation To define the most suitable condition for adsorption the of RB has been reported in the literature, the removal of effect of several parameters on this process was checked.

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For studying the effect of solution pH on dye adsorption, Samples of H. communis sponge skeleton before and after experiments at different pH values (ranging from 2to the adsorption process were observed using a Keyence BZ- 11) were performed for an initial dye concentration of 9000 (Japan) microscope in light as well as in fluorescent 100 mg/L. To observe the effect of NaCl on dye adsorption, microscopy mode. different amounts of salt (in concentrations from 0.01 Photographs were also taken using an EVO40 to 1.0 M) were used with 100 mg/L of CuPC solution scanning electron microscope (Zeiss, Germany) to obtain at initial pH. An adsorbent dose of 0.1 g, contact time data on surface morphology and structure. 60 min, initial pH and no NaCl were used for all of the above experiments. The desorption efficiencies of CuPC from the sorbent 2.4 Photocatalytic activity were measured after the adsorption experiments were completed (100 mg/L, pH=2, contact time 120 min). The catalytic reactions were carried out by adding 20 mg These studies were carried out in water at ambient of a photocatalyst (sponge skeleton with CuPC, obtained temperature and in water with ultrasound at 40, 50 after adsorption from 400 mg/L solution at pH=2 for 4 h) and 60 °C, for 300 min. A standard technique was to a glass tube reactor containing 30 mL of 10 mg/L RB used to determine the dye concentration using a solution. The reaction mixture was continuously shaken UV-Vis spectrophotometer (Jasco V750, Japan). by a magnetic stirrer. Photochemical degradation was carried out in a UV-reactor system (UV-RS-2, Heareus, Germany), equipped with a 150 W medium-pressure 2.3 Analysis mercury lamp, surrounded by a water-cooling quartz jacket to cool the lamp. At given intervals of illumination, Cross polarization solid state magic angle spinning 1 mL of the RB solution was taken and decreases in the nuclear magnetic resonance (13C CP MAS NMR) spectra concentration of dye were analyzed by a Jasco V750 UV- were obtained at a 13C frequency of 100.63 MHz on a DSX Vis spectrophotometer (halogen lamp) at λ=554 nm. The spectrometer (Bruker, Germany). Samples were packed in efficiency of degradation catalyzed by marine sponge a 4-mm-diameter cylindrical zirconia rotor and spun at skeleton–CuPC hybrid material (CuPC-HcS) was evaluated 8 kHz. by means of the RB degradation ratio, given by the To confirm the effectiveness of the adsorption following formula: process, Fourier transform infrared spectroscopy

(FTIR) was performed, using a Vertex 70 spectrometer [(C0 − C)/C0] × 100% (3) (Bruker, Germany). The samples were analyzed in the form of tablets, produced by pressing a mixture where C and C0 represent the time-dependent concentration of anhydrous KBr (ca. 0.25 g) and 1 mg of the tested and the initial concentration (determined from the substance in a special steel ring under a pressure of calibration curve), respectively. All the experiments were 10 MPa. The investigation was performed over a performed three times. wavenumber range of 4000–400 cm-1 at a resolution of 0.5 cm-1. Energy dispersive spectroscopy (EDS) measurements 3 Results and discussion were carried out using a PTG Prism Si(Li) (Princeton Gamma Tech., USA) energy-dispersive X-ray spectrometer. Before the analysis, samples were placed on the ground 3.1 Adsorption and desorption process with a carbon paste or tape. The presence of carbon materials is needed to create a conductive layer which 3.1.1 Influence of time and initial dye concentration provides the delivery of electric charge from the sample. Thermogravimetric (TG) analysis of samples of The influence of time and initial dye concentration was products was carried out with a Jupiter STA 449F3 investigated in optimal experimental conditions, with analyzer (Netzsch, Germany) with an Al2O3 crucible. The controlled pH, ionic strength and temperature. measurements were performed in a nitrogen atmosphere The changes in the quantity of dye adsorbed (qt) at a heating rate of 10 °C·min-1, over a temperature range of are significant in the first minutes of the process; then 25–1000 °C, with an initial sample weight of approximately with time they become smaller until equilibrium is 5 mg. reached (Fig. 1). Equilibrium is reached faster for a CuPC

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results were obtained in the presence of 1M NaCl in the dye solution. The addition of salt ions affects the interaction between adsorbent and adsorbate, due to the increase in ionic strength, a decrease in the thickness of the electrical double layer surrounding particles in solution is observed. Both the repulsive and attractive interactions will be weaker, which increases or decreases the efficiency of the process, depending on which of these interactions occur between the particles in solution [39]. Furthermore, the addition of NaCl increases the value of van der Waals forces, ion- ion and ion-dipole forces, which positively influences the effectiveness of the adsorption process [40]. Desorption tests were performed to evaluate the strength of the connection between H. communis skeleton Figure 1: Adsorption capacity of CuPC on H. communis sponge and dye. It was observed that shaking with water at room skeleton, as a function of time (results obtained at pH=2). temperature and ultrasound-assisted washing at 30, 50 and 60 °C, causes only slight elution of the previously solution with lower initial concentration (60 min for dye adsorbed CuPC (5%). concentrations of 50 and 100 mg/L and 300 min for 200 mg/L). The adsorption efficiency reaches 100% for CuPC (independently of initial dye concentration). The quantity 3.2 Kinetics and isotherms of adsorption of dye adsorbed on the support increases when the initial dye concentration increases, which is a typical situation To determine the rate and mechanism of the process explained by the influence of mass gradient and the action of adsorption, pseudo-first-order and pseudo-second- of mass transfer driving forces [37]. order kinetic models were fitted to the experimental data. These models present the correlations between changes in the concentration of adsorbate as 3.1.2 Influence of pH and ionic strength a function of the time for which the adsorption process is continued, until equilibrium is reached. The next stage of the study involved examination of The linear forms of the kinetic models of pseudo-first different adsorption conditions (pH and ionic strength) and pseudo-second order are given and described in detail and their influence on process efficiency. In each case in [41] and [42]. only one parameter was changed, while all other variables As can be seen from Fig. 2a and Table 1, the pseudo- (time, initial concentration) were kept constant. second-order model better describes the experimental In the case of CuPC the greatest effect on the quantity of data. In spite of the relatively high values of the correlation dye adsorbed (qt) resulted from the pH of the dye solution. coefficients for the pseudo-first-order model, it cannot be Under basic and weak acidic conditions, the values obtained considered useful because of the large differences between for the efficiency of the process are similar, at around 60%, the experimental and calculated sorption capacities. but a considerable change is noted at pH=2, where the The adsorption isotherm parameters were calculated efficiency increases to 98%. In these conditions ionization according to the Freundlich (4) [43] and Langmuir (5) [44] of functional groups of the amino acids building spongin models: + (NH2→NH3 ) takes place, which results in their mutual attraction (in contrast to the repulsive forces in an alkaline (4) environment). These results suggest that the driving force of the adsorption may be electrostatic interactions [38]. The dye molecules can reach the surface of the H. communis where Ce denotes the equilibrium concentration of the sponge skeleton, where they can interact via hydrogen dye solution (mg/L), qe is the quantity of dye adsorbed at bonding. The pH=2 was chosen as the best condition for all equilibrium (mg/g), Kf (mg/g) and n are the Freundlich further adsorption process. constants. The values of Kf and n can be determined from

The adsorption of dye on the marine sponge skeleton the intercept and gradient of the plot of log(qe) against was positively affected by the presence of NaCl. The best log(Ce).

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Figure 2: Pseudo-second-order (a) model fit for adsorption of CuPC onH. communis skeleton and fit of the experimental data to the Langmuir and Freundlich models (b).

Table 1: Kinetic parameters of CuPC adsorption on H. communis the adsorption of CuPC on marine sponge skeleton is a skeleton. complex process. The calculated correlation coefficients (r2) are similar, 0.917 for Langmuir and 0.957 for Parameters Units 50 mg/L 100 mg/L 200 mg/L Freundlich model. H. communis sponge skeleton has a q mg/g 12.50 24.98 48.93 e,exp relatively high maximum adsorption capacity (qm) for Pseudo-first-order copper phthalocyanine (83.36 mg/g). The parameter n in r - 0.988 0.811 0.933 the Freundlich isotherm model is equal to 3.46 (n>1). A value of 1/n below one indicates normal adsorption, while k1 1/min 0.034 0.020 0.012 if 1/n is above one it indicates cooperative adsorption; q mg/g 5.48 7.21 30.41 e,cal additionally, the smaller the value of 1/n, the greater the Pseudo-second-order heterogeneity of the adsorbate. r - 0.999 0.999 0.992 k2 1/min 0.021 0.009 0.001 3.3 13C CP/MAS NMR qe,cal mg/g 12.66 25.32 50.65 h mg/g min 3.32 5.88 2.83 Figure 3 shows the results of 13C MAS NMR analysis. Marine sponge spongin has an inexact chemical structure, where: k1 and k2 - rate constants of the pseudo-ﬁrst-order and pseudo-second-order models respectively, r - correlation coefficient, but tentative assignments of some of the unknown components can be made. Signals in the range 17.7–69.6 qe,exp and qe,cal - experimental and calculated adsorption capacity at equilibrium, h - initial adsorption rate. ppm suggest the presence of alkyl carbons (RCH2, RCH3),

carbons bonded to oxygen (RCH2O–) (above 50 ppm),

nitrogen (RCH2NH2), as well as halogen atoms (C–Cl, C–Br, (5) C–I, C–SR) (below 50 ppm). The peaks above 115 ppm can be assigned to RHC=CH and aromatic carbons, and those where Ce is the equilibrium concentration of the dye at 170.0 and 172.1 to C=O in acids and esters respectively. solution (mg/L), qm is the maximum adsorption capacity As stated in [45,46] the signals observed in the CuPC (mg/g), and b is the Langmuir constant (L/mg), which is spectrum can be ascribed to C1 (141.3 ppm), C2 (128.4 calculated from the intercept and downward linear slope ppm), C3 (126.2 ppm) and C4 (138.4 ppm) incorporated in of the graphs of Ce/qe and Ce. the carbon structure. Comparing the carbon NMR spectra

A graph of qe against Ce for the adsorption isotherms of marine sponge skeleton with copper phthalocyanine of CuPC on marine sponge skeletons is shown in Fig. 2b. (Fig. 3b and 3c) with the spectra of a sample of H. Analysis of the isotherm parameters obtained for communis (Fig. 3d) and CuPC (Fig. 3a), it is evident that the Freundlich and Langmuir models indicates that the CuPC macrocycle carbons appear as a series of lines

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the H. communis spectrum. The most important changes related to the stretching asymmetric vibrations of sulfonic - + acid salts (–SO3 M ) occurring in the wavenumber range 1250–1140 cm-1 in the form of a broad band divided into a few smaller peaks. The position of these signals is strongly dependent on the nature of the metal ion which - is incorporated in the –SO3 group, as reported earlier in [48]. The effect of the type of carbon atom (alkyl or aryl) connected to the sulfur atom on the position and shape of these bands is much less significant. Additionally, it should be noted that an additional signal is present in the spectrum of the CuPC-HcS material at 1026 cm-1. This peak is shifted in comparison with the spectrum of the crude dye (maximum at 1032 cm-1), which proves the effective adsorption of the CuPC [13]. Furthermore, a signal confirming deposition of the dye, absent from the spectrum of H. communis, is present at 1339 cm-1, generated by stretching vibrations of the –C=C–N– bonds in indole rings. In the CuPC-HcS spectrum a series of specific bands are observed at wavenumbers 746, 699, 649 and 599 cm-1, which come from the interactions between Cu and its ligands. Additionally the broad and strong signal in the range 3500–3200 cm-1 in this spectrum can be attributed to stretching vibrations of –OH and –NH groups. This signal has a different shape than in the spectra of the materials before the adsorption process. A slight shift in the maximum of this band suggests the formation of chemical bonds (hydrogen bonds) between the sponge and the phthalocyanine dye.

3.5 EDS Figure 3: 13C NMR spectra of CuPC (a), sample 1 (pH=2, 120 min, 500 mg/L) (b), sample 2 (pH=2, 120 min, 400 mg/L) (c), H. communis Table 2 shows the results of energy dispersive spectroscopy skeleton (d) and chemical structure of copper phthalocyanine- (EDS) analysis for H. communis skeleton, copper 3,4′,4″,4″′-tetrasulfonic acid tetrasodium salt (CuPC). phthalocyanine and selected samples obtained after the adsorption process (sample 1: pH=2, 120 min, 500 mg/L; in the range 120–130 ppm in the spectra of samples 1 and sample 2: pH=2, 120 min, 400 mg/L; sample 3: pH=2, 2. Furthermore a slight downﬁeld shift movement of the 120 min, 300 mg/L). alkyl carbons of the marine sponge skeleton provides The H. communis skeleton, as well as CuPC, consists additional characterization of the new materials. mainly of carbon and nitrogen, but these elements are not shown in the table. The data given in Table 2 indicate the presence of oxygen, sodium, aluminum, silica, sulfur, 3.4 FTIR chlorine, iodine and calcium. These elements are naturally occurring in the H. communis skeleton. The occurrence The results of FTIR analysis of marine sponge skeleton of elements at low concentration (I, Cl, S, Ca, Al) is in prior to and after the adsorption process, and of CuPC, are accordance with previously published reports [49,50] and presented in Fig. 4. The detailed characterization of the H. implies that some of the spongin-built amino acids may communis skeleton can be found in our previous work [47]. contain heteroatoms. However, the chemical composition Many differences appear in the spectrum after dye of spongin has not yet been precisely determined. In adsorption, especially below 1200 cm-1, compared with the measured region of the samples after the adsorption

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Table 2: Quantiﬁcation of elements in the analyzed marine sponge skeleton, copper phthalocyanine and selected samples.

Element content (wt.%) H. communis CuPC SAMPLE 1 SAMPLE 2 SAMPLE 3 O 68.42 22.06 74.77 73.48 68.48 Na 0.99 26.18 1.70 1.06 1.07 Al 6.12 0.59 3.65 3.99 4.97 Si 6.98 1.03 1.41 5.11 2.28 S 5.91 31.45 8.63 9.50 11.97 Cl 0.96 6.81 5.63 5.07 7.09 Cu - 11.88 0.31 0.28 0.19 K - - 2.31 1.45 3.41 I 9.20 - 1.51 0.04 0.28 Ca 1.42 - 0.09 0.03 0.27 Total 100.00 100.00 100.00 100.00 100.00

Figure 4: FTIR spectra of the purified H. communis skeleton, CuPC and dye adsorbed onto sponge skeleton (pH=2, 120 min, 400 mg/L), in different wavenumber ranges. process we observe the presence of Cu atoms, which provides direct confirmation of the effectiveness of the adsorption process.

3.6 TG

Figure 5 shows TG curves for the H. communis skeleton (HcS), copper phthalocyanine tetrasulfonic acid (CuPC), and the obtained hybrid material (CuPC-HcS). Thermal decomposition of sulfonated phthalocyanine proceeds in a few steps. The first slight mass loss (about 10%) is probably connected with loss of moisture (up to 200 °C). Between 430 and 530 °C, the material had lost another 10% of its mass, the loss and fragmentation of substituent Figure 5: TG curves of thermal decomposition of the H. communis units of the environment of the phthalocyanine molecule skeleton, copper phthalocyanine and selected sample obtained occurs [51]. Studies [52] have found that in the range after the adsorption process (pH=2, 120 min, 400 mg/L).

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Figure 6: Images of H. communis fibrous skeleton (a) and CuPC-HcS (b) from fluorescent and light microscopes.

530–730 °C rupture of macroheterocycles takes place. visible especially in the case of red light, which provides The residues remaining after thermal decomposition indirect confirmation that the adsorption is a chemical correspond to the metal (in this case copper) oxides [53]. process (Fig. 6). The rate of degradation of marine sponge skeleton is Fluorescence of some other Demospongiae species higher than that of CuPC. This is a result of the thermal have been reported, but the measurements were made decomposition of spongin. As observed on the TG curve, in situ and fluorescence was caused by symbiotic or the obtained hybrid material undergoes two stages of commensal algae or cyanobacteria on the sponge skeletons mass loss: the first below 120 °C, and the second starting [55]. at 230 °C to reach a plateau near 560 °C. These can be Although the photographs from the light microscope attributed respectively to water loss and degradation of the imply that the spongin fibers are entirely covered, the protein-like support. The total mass loss in the case of HcS SEM images reveal that there is not a homogenous layer alone (73%) is higher than in the case of CuPC-HcS (67%). of CuPC on the fibers. The bundle of fibrils forming the spongin fibers is still visible (Fig. 7).

3.7 Microscopy observations 3.8 Catalysis The images from light, fluorescent and SEM microscopes of samples before and after the adsorption process are Electro- and sonochemical reactions or oxidation by presented below. The adsorption process conditions were ozone are sometimes used to remove Rhodamine B, but as follows: pH=2, 120 min, 400 mg/L. the most frequently used methods are adsorption and It is reported in the literature [54] that autofluorescence photodegradation. Very often porphyrin or phthalocyanines is usually a superposition of fluorescence from a mixture are used as catalysts in these processes. The photodegradation of individual fluorescent molecules (fluorophores). process is very often enhanced by using an externaloxidant, Fluorophores such as amino acids (e.g. tyrosine) emit e.g. hydrogen peroxide [56-58]. fluorescent light due to heterocyclic aromatic rings (such To investigate the catalytic performance of the prepared structures are also present in spongin) or conjugated double heterogeneous catalysts and the influence of various factors bonds within their molecular structures. H. communis on RB elimination, a number of experiments were carried fibers exhibit photoluminescence, in blue, green as well out. We monitored the photodegradation of RB molecules as red light. A decrease in fluorescent intensity is clearly under UV irradiation (190–300 nm) by measuring the UV-

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Figure 7: SEM of H. communis prior to (a) and after (b), (c) the adsorption process at different magnifications.

Vis spectra of filtrate solutions. H. communis skeleton is capable of adsorbing dyes, as was demonstrated above, thus it is possible that RB will also be adsorbed on this support. According to the results presented in Fig. 8 (curve b), the adsorption of Rhodamine B on the sponge skeleton does indeed take place, and the efficiency of the process reaches 32%. This parameter is even higher (65%) when CuPC+HcS (obtained from dye solution at concentration of 400 mg/L at pH=2 for 4 h) is used as the adsorbent (Fig. 8, curve d). A possible explanation of this effect (that sponge with previously adsorbed dye adsorbs better than the pure spongin) may be that functional groups of CuPC interact with the cationic dye RB by electrostatic interaction. UV irradiation itself does not have any influence on Figure 8: Effect of time on adsorption and photocatalytic RB degradation (Fig. 8, curve a). The effect is observed degradation of RB under different conditions: (a) UV irradiation; (b) adsorption on sponge skeleton in visible light; (c) H O in UV; in the presence of H2O2 (62%; Fig. 8, curve c) and CuPC- 2 2 HcS (75%; Fig. 8, curve e). As expected, the addition of (d) adsorption on CuPC-HcS in visible light; (e) CuPC-HcS in UV; (f)

CuPC-HcS and H2O2 in UV. H2O2 improves the efficiency of decolorization, but is not effective enough to oxidize RB if used alone. Up to 95% of the RB was photodegraded when both CuPC-HcS as catalyst reactive oxygen species via the corresponding propagation and H2O2 as oxidant were present, and the concentration reactions (V–VII): of RB decreased quickly within 1 h (Fig. 8, curve f).

A possible mechanism of dye degradation H2O2 + hv → 2OH• (IV) under irradiation is proposed based on the reported mechanism involving both H2O2 and CuPC. The action H2O2 + OH• → HO2• + H2O (V) of metalphthalocyanine causes several successive reactions with the excited singlet and triplet form H2O2 + HO2• → OH• + O2 + H2O (VI) of metalphthalocyanine and oxygen, which finally decompose the dye [13,59]: 2 OH2• → H2O2 + O2 (VII)

1 * 3 * MPc + hv → MPc → MPc (I) Moreover CuPC can act as a catalyst and activate the H2O2 molecules: 1 * 3 1 * MPc + O2 → O2 + MPc (II) II III – Cu Pc + H2O2 → Cu Pc + OH + OH• (VIII) 1 * O2 + dye → degradation product (III) As stated in [62] there is a possibility for copper to exist

According to [60,61], simultaneously the photolysis of H2O2 as Cu(III) in a complex with porphyrin. The produced generates the hydroxyl radical (equation IV) and other hydroxyl radical oxidizes RB:

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RB + OH• → [RB(OH•)] (IX) contrast, in our study we used a biopolymer matrix which is inert under UV irradiation. Hence the presented results [RB(OH•)] → degradation product (X) relate only to the activity of the phthalocyanine in the dye decomposition process. RB + hv → RB* (XI)

* + – RB + O2 → RB • + O2 • (XII) 4 Conclusions

+ RB • + O2 → degradation product (XIII) In this study a copper phthalocyanine–H. communis marine sponge skeleton hybrid material (CuPC-HcS) was The hydroxyl radical generated from hydrogen peroxide produced with high efficiency by an adsorption process. reacts with RB (equation IX) and gives an intermediate Adsorption is a fast and simple way to obtain such systems, product, which decomposes into the final oxidation and does not require sophisticated equipment. The product (X). Furthermore, irradiation with UV light causes highest efficiencies were obtained from an acidic solution, the excitation of rhodamine (XI). The produced RB* reacts which suggests that electrostatic interactions occur with oxygen, and as a result the cationic radical form of between the dyes and support. Furthermore, results from + – the dye (RB •) and superoxide (O2 •) are formed (XII). spectroscopic analysis indicate the formation of hydrogen Subsequently, via further reaction with oxygen, the dye bonds. The product was used in the photobleaching is degraded [14,63]. The oxidation products of RB are of Rhodamine B solution. In these reactions CuPC-HcS thoroughly investigated and described in [64,65]. serves as a photosensitizer and H2O2 as an external Zhang et al. investigated the photocatalytic activities oxidant. Adsorption and simultaneous catalytic oxidation of 2,9,16,23-tetra-nitrophthalocyanine copper(II) are responsible for RB removal, and all of the analyses

(TNCuPc)/TiO2 composites with respect to Rhodamine B performed suggest that it is highly desirable to integrate degradation. The TiO2 nanofibers function as an electron the advantages of both high adsorption capacity and trap for the excited surface-adsorbed TNCuPc dye. The photocatalytic activity into a single compound. trapped electron subsequently induces the generation of active oxygen species, which degrade Rhodamine B with Acknowledgments: This work was financially supported high efficiency, up to almost 90% [57]. Shang et al. also by Poznan University of Technology research grant no. showed the effect of copper phthalocyanine tetrasulfonic 03/32/DSMK/0610 (Poland) as well as by DFG Grant HE acid-sensitized TiO2 on Rhodamine B degradation [58]. 394/3-2 (Germany). The authors achieved total degradation efficiency, although the process required the hydrothermal synthesis of the catalyst, which increases operational costs in References comparison with the fast and facile adsorption technique described in the present work. The authors confirmed [1] Sevim A. M., Ilgün C., Gül A., Preparation of heterogeneous that the presence of copper phthalocyanine considerably phthalocyanine catalysts by cotton fabric dyeing, Dyes enhanced the photocatalytic activity of titanium dioxide. Pigments, 2011, 89, 162–168 Another study on the synthesis of a titanium dioxide– [2] Paul S., Joseph M., Polypyrrole functionalized with FePcTSA for NO sensor application, Sensors Actuators B Chem., 2009, 140, copper phthalocyanine hybrid composite and its use in 2 439–444 decomposition of Rhodamine B under UV irradiation was [3] Singh V. K., Kanaparthi R. K., Giribabu L., Emerging molecular published by Mekprasart et al. The system obtained cause design strategies of unsymmetrical phthalocyanines for decomposition of over 80% of the dye. The significant dye-sensitized solar cell applications, RSC Adv., 2014, 4, improvement in photocatalytic activity in the case of the 6970–6984 hybrid composite may be linked to the enhancement of [4] Asedegbega-Nieto E., Pérez-Cadenas M., Carter J., Anderson J. optical absorption and the inhibition of electron–hole A., Guerrero-Ruiz A., Preparation and surface functionalization of MWCNTs: study of the composite materials produced by the recombination caused by the presence of CuPC in the TiO 2 interaction with an iron phthalocyanine complex, Nanoscale matrix [66]. Res. Lett., 2011, 6, 1–4 In the cited works, copper phthalocyanines were [5] Harbeck M., Erbahar D. D., Gürol I., Musluoğlu E., Ahsen V., Öztürk Z. Z., Phthalocyanines as sensitive coatings for QCM immobilized on TiO2, which itself is photocatalytically active. Thus the results obtained represent a combination sensors operating in liquids for the detection of organic compounds, Sensors Actuators B Chem., 2010, 150, 346–354 of the photocatalytic properties of both materials. By

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Journal of Hazardous Materials 347 (2018) 78–88

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Iron(III) phthalocyanine supported on a spongin scaffold as an

advanced photocatalyst in a highly efﬁcient removal process of

halophenols and bisphenol A

a a b

Małgorzata Norman , Sonia Zółtowska-Aksamitowska˙ , Agnieszka Zgoła-Grzeskowiak´ ,

c a,∗

Hermann Ehrlich , Teoﬁl Jesionowski

Poznan University of Technology, Faculty of Chemical Technology, Institute of Chemical Technology and Engineering, Berdychowo 4, 60965, Poznan, Poland

Poznan University of Technology, Faculty of Chemical Technology, Institute of Chemistry and Technical Electrochemistry, Berdychowo 4, 60965, Poznan, Poland

TU Bergakademie Freiberg, Institute of Experimental Physics, Leipziger 23, 09599, Freiberg, Germany

h i g h l i g h t s g r a p h i c a l a b s t r a c t

•

Supported iron(III) phthalocyanine as

photocatalyst in advanced oxidation process.

•

Combined procedure for phe-

nol, halophenols and bisphenol A degradation.

•

Synergistic effect of catalyst, hydro-

gen peroxide, UV irradiation and

adsorption.

a r t i c l e i n f o a b s t r a c t

Article history: This study investigated for the ﬁrst time the degradation of phenol, chlorophenol, ﬂuorophenol and

Received 27 September 2017

bisphenol A (BPA) by the novel iron phthalocyanine/spongin hybrid material under various process con-

Received in revised form 6 December 2017

ditions: hydrogen peroxide and UV irradiation. The heterogeneous catalyst, iron phthalocyanine/spongin

Accepted 20 December 2017

(SFe), was produced by an adsorption process. The product obtained was investigated by a variety of spec-

Available online 23 December 2017

troscopic techniques – X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray spectroscopy

(EDS), Fourier transform infrared spectroscopy (FTIR) and carbon-13 nuclear magnetic resonance ( C

Keywords:

NMR) – as well as elemental and thermal analysis. The study conﬁrmed the stable immobilization of

Hippospongia communis

Spongin the dye on the biopolymer. The results demonstrate that the degradation of phenols and BPA followed

pseudo-second-order kinetics under different experimental conditions. The synergy of SFe, H2O2 and UV

Advanced oxidation process

Halphenol and bisphenol A removal was found to produce a signiﬁcant increase in the removal efﬁciency and resulted in complete removal

Photodegradation kinetics of contaminants in a short time of 1 h. The reaction products were identiﬁed by high-performance liq-

uid chromatography/mass spectrometry (HPLC-MS) and possible degradation pathways were proposed,

featuring a series of steps including cleavage of C C bonds and oxidation.

∗

Corresponding author.

E-mail address: teoﬁ[email protected] (T. Jesionowski).

https://doi.org/10.1016/j.jhazmat.2017.12.055

M. Norman et al. / Journal of Hazardous Materials 347 (2018) 78–88 79

1. Introduction materials, such as polycarbonate, epoxy resins, ﬂame retardants

and plasticizers [31,32]. Due to their high production volumes

Over the last few decades, chemical oxidation processes such and widespread application, phenols and their derivatives are

as ozonation, wet oxidation, hydrogen peroxide-based methods released into the environment from industrial and municipal liq-

and electrochemical or photochemical oxidation have been studied uids wastes. This is particularly undesirable since phenols are

extensively as means for the decontamination of water containing considered hematotoxic and hepatotoxic, and trigger mutagene-

various organic pollutants [1,2]. In particular, the applicability of sis and carcinogenesis in humans and other living organisms [33].

heterogeneous photocatalytic processes has received considerable Concomitantly, many studies have conﬁrmed that BPA causes liver

attention [3]. The use of a heterogeneous catalyst may overcome damage, reproductive and developmental toxicity, diabetes risk,

the drawbacks of homogenous systems. It causes an increase in breast cancer, cardiovascular disease and endocrine system disrup-

surface area and hence better access to surface active sites, usually tion [34].

improves the thermal, hydrolytic and chemical stability of the cat- High toxicity, resistance to natural degradation and harmful-

alytic material, and enables its recyclability. Moreover, almost all ness to the environment contribute to the leading position of the

industrial catalytic processes use heterogeneous catalysts in view aforementioned substances among priority control pollutants [35].

of their main advantage: ease of separation from the reaction prod- Several methods have been developed for the removal of these pol-

uct. Recently, among the commonly used supports, researchers lutants from aqueous solutions, including biochemical oxidation,

have shown increased interest in those of renewable natural origin. adsorption, electrochemical oxidation, wet chemical oxidation,

The conditions during the photocatalytic reaction are mild: ozonation, sonochemical oxidation and biodegradation [36–40].

increased temperature or pressure is not necessary. The use of In the present study, 3D spongin scaffolds isolated from Hip-

synthetic supports requires their prior preparation and/or synthe- pospongia communis demosponge skeletons were used for the ﬁrst

sis, which would involve unwanted costs and additional reagents, time as a support for iron phthalocyanine adsorption. The inter-

byproducts or solvents. The use of materials of natural origin actions between ﬁbrous spongin and iron phthalocyanine were

removes this inconvenience. Recent trends toward sustainable explored using multiple analytical techniques. This work aims to

development emphasize the importance of renewable materials analyze the photocatalytic activity of the obtained system in the

and biopolymers as catalyst supports. degradation of phenolic pollutants. The effect of H2O2 and UV on

In the photodegradation of dyes, phenols, pesticides and other the degradation rate of phenol, chlorophenol, ﬂuorophenol and

organic pollutants, such biopolymers as chitosan [4,5], cellulose BPA by iron phthalocyanine/spongin photocatalysis was examined,

[6–8] and collagen [9,10] have previously been used. However, and the intermediates formed in the degradation process were

there are few reports regarding the application of these supports identiﬁed. Finally, a degradation mechanism for halophenols and

with phthalocyanine as a catalyst for photodegradation [11–13]. bisphenol A was proposed.

Natural origin matrices/supports are of industrial interest due

to their strong absorption capabilities, low toxicity and versatile 2. Experimental

biotechnological and biocompatible properties.

Spongin, a structurally mechanically stable protein of sponge 2.1. Reagents

origin [14], meets the requirements for an ecofriendly and cost-

′ ′′ ′′′

effective support. Recently, spongin-based scaffolds have been used Iron(III) phthalocyanine-4,4 ,4 ,4 -tetrasulfonic acid, the com-

for diverse applications in bioinspired materials science [15–17] pound with oxygen monosodium salt hydrate (denoted as FePC)

including extreme biomimetics [18,19]. Due to the internation- (Sigma–Aldrich) and other chemicals were of analytical grade and

ally developed marine ranching of spongin-based bath sponges, were used as received from the suppliers without further puriﬁca-

this biological material represents a renewable source with real tion.

prospects of practical industrial-scale applications [20]. Perhaps the HPLC-grade methanol and tandards of bisphenol A, phe-

most signiﬁcant feature, and one which also makes it suitable as a nol, 4-chlorophenol and 4-ﬂuorophenol were obtained from

metal phthalocyanine support, is its resistance to acids, as well as Sigma–Aldrich. HPLC-grade water was prepared by reverse osmo-

its unique ﬁbrous morphology, microporosity and material proper- sis in a Demiwa system (Watek), followed by double distillation

ties [21]. The three-dimensional, reticular structure of spongin can from a quartz apparatus.

be expected to give reactants effective access to the catalyst.

Phenols, olefins, aromatics, dyes and sulfides can be efficiently 2.2. Hybrid material preparation and characterization

oxidized to valuable products by iron phthalocyanine, alone or

immobilized on a support [22]. This compound is extensively used Hippospongia communis (Demospongiae: Porifera) marine

as a catalyst in photochemical degradation (in the presence of sponge skeletons obtained from INTIB GmbH were used for the

light and H2O2), photocatalysis (light) [23] and chemical oxidation preparation of spongin scaffolds (Fig. 1) according to the proce-

processes (H2O2) [24–26]. Numerous studies have attempted to dure described in Supplementary material and in our previous work

explain the catalytic activity of metal phthalocyanines with respect [41]. 0.1 g of spongin scaffold fragment was added to 25 mL of iron

to organic compounds. During irradiation in visible light they are phthalocyanine water solution (concentration 100–600 mg/L, pH

excited to the singlet state (1 MPc)* and afterwards converted to 2). The mixture was placed in a ﬂask and stirred at room tempera-

the triplet state (3 MPc)*. This excited triplet state then interacts ture. After 6 h, the obtained samples were washed with distilled

with the ground state of molecular oxygen to generate excited sin- water under ultrasound and then dried. The fabricated samples

glet oxygen [27]. If hydrogen peroxide is involved in the reaction used for photocatalytic studies were obtained from 400, 500 and

system, some additional reactions occur. Visible radiation leads 600 mg/L solution and denoted SFe 400, SFe 500 and SFe 600 respec-

•

to the formation and reaction of HO radicals with high oxidative tively.

activity and/or formation of a nucleophilic iron(III) peroxo complex

[HOOFeIII (PcS)] [28]. 2.2.1. X-ray photoelectron spectroscopy (XPS)

Phenol and its derivatives have been used as components of Studies on the composition and chemical state of selected ele-

dyes, polymers, drugs and other organic substances [29,30]. Bisphe- ments were made by X-ray photoelectron spectroscopy using a

nol A is present in household and commercial products as an VSW apparatus (Vacuum Systems Workshop Ltd.) in which the X-

important component for the production of a variety of chemical ray source was operated with an Mg anode (K␣: 1253.6 eV). The XPS

80 M. Norman et al. / Journal of Hazardous Materials 347 (2018) 78–88

Fig. 1. Spongin-based scaffold (A) isolated from H. communis marine sponge skeletons has a well-developed microporous and ﬁbrous 3D architecture (SEM image, B).

Table 1

Results of elemental and EDS analysis (SFe 400 spongin/iron phthalocyanine mate-

rial obtained in a time of 5 h, at pH = 2 with 400 mg/L of FePC solution).

Element (%) Spongin FePC SFe 400

Elemental analysis

C 40.11 35.41 42.19

N 13.57 11.29 13.31

H 6.19 2.51 6.33

S 1.90 9.67 1.71

EDS

C 85.37 89.04 85.57

N 2.48 – 0.31

O 9.56 1.84 8.82

Na 0.10 1.33 0.01

K 0.03 – 0.03

Mg 0.08 – 0.04

Ca 0.07 – 0.04

Al 0.48 0.05 0.58

Si 0.34 0.04 0.56

S 0.55 5.29 1.46

Fe 0.09 2.41 0.52

Fig. 2. XPS survey scan of spongin, iron phthalocyanine and the obtained material

P 0.03 – 0.03

SFE 400.

Cl 0.21 – 1.33

I 0.61 – 0.70

−8 Total 100.00 100.00 100.00

analysis chamber has a base vacuum of 3 × 10 mbar. The spectra

were evaluated using the program XPSPEAK 4.1.

2.2.5. Thermal analysis (TG)

2.2.2. Energy-dispersive X-ray spectroscopy (EDS)

TG analyses of the samples were carried out in dynamic condi-

The chemical composition of the samples was analyzed using

tions using an STA 449 F3 Jupiter apparatus (Netzsch) in a nitrogen

a PTG Prism Si(Li) (Princeton Gamma Tech.,) energy-dispersive X- ◦

atmosphere, at a heating rate of 10 C/min over the temperature

ray spectrometer. Before the analysis, samples were placed on the ◦

range 25–1000 C. Approximately 5 mg of the samples were placed

ground with a carbon paste or tape. The presence of carbon mate-

in open Al2O3crucibles.

rials is needed to create a conductive layer, which provides the

Moreover, the Fourier transform infrared spectroscopy (FTIR)

delivery of electric charge from the sample.

were also performed. The procedure is described in Supplementary

material.

2.2.3. Elemental analysis

A Vario El Cube instrument (Elementar Analysensysteme

2.3. Catalysis investigation

GmbH) was used for elemental analysis. The operating principle

of the device is based on burning a sample in ﬂowing oxygen at

2.3.1. Photocatalytic test

◦

temperatures of up to 1200 C. After passing through appropriate

The photodegradation experiments were conducted using a

catalysts in a helium stream, the resulting gases were separated

UV-RS-2 photocatalytic reaction instrument (Heareus): an inter-

in an adsorption column and then recorded using a detector. The

nal mercury lamp equipped with a cut-off glass ﬁlter transmitting

measurements were made three times, with a measurement error

>150 nm. The system was cooled by circulating water running

of 0.001%.

through an interlayer, and magnetic agitation was used so that

the catalyst could be stirred in the reaction solution. For a typi-

2.2.4. Carbon-13 nuclear magnetic resonance ( C NMR)

cal experiment, 30 mL of a 2 mg/L solution of phenol, chlorophenol,

For Cross Polarization Magic Angle Spinning Nuclear Magnetic ﬂuorophenol or BPA was mixed with 1 ml 30% hydrogen peroxide

Resonance ( C CP/MAS NMR) the spectrometer used was a Bruker and SFe 400 catalyst (0.04 g) (marine sponge skeleton with FePC,

DSX (Bruker) with MAS probes (for 100 mg of sample) with ZrO2 obtained after adsorption from a 400 mg/L solution at pH = 2 for

rotors of 4 mm in diameter. Rotation at 8 kHz enabled better reso- 5 h). Each system was irradiated for 60 min, and every 10 min 1 mL

lution of inequivalent sites and better quantiﬁcation of sites. of the investigated solution was sampled and measured chromato-

M. Norman et al. / Journal of Hazardous Materials 347 (2018) 78–88 81

Fig. 3. C NMR spectra of spongin and a selected sample obtained after adsorption from 400 mg/L and 500 mg/L FePC solution.

graphically. The same experiment was also carried out only in the 2.3.4. Method performance

presence of UV light and hydrogen peroxide (1 mL of 30% H2O2). The linearity of the method was tested over a wide range for all

The synergy index (SI) of photooxidation was calculated using analytes. The instrumental limit of detection (LOD) and the instru-

Eq. (1): mental limit of quantitation (LOQ) were calculated on the basis of

the signal-to-noise (S/N) ratio: S/N = 3 for calculation of LOD and

SI = (1) S/N = 10 for calculation of LOQ. Precision and accuracy were not

E1 + E2 + E3 + E4

tested, because sample preparation included only a dilution step.

where E is the percentage efﬁciency when UV light, H2O2 and SFe Therefore, only the injection precision of the instrument applies in

400 were used altogether, and E with subscripts represents efﬁ- this procedure, which was always below 1%.

ciency under different process conditions (adsorption, UV light,

H2O2, SFe 400). SI > 1 indicates that the combined process has a

3. Results and discussion

positive synergic effect, while SI < 1 represents a negative effect

[42].

The spongin/iron phthalocyanine hybrid material was pro-

duced by an adsorption process (see Supplementary material).

2.3.2. HPLC-FD analysis

This method has the advantages of fast, low-cost, green synthesis

For calculation of degradation efﬁciency, the solution after pho-

without the use of any additional hazardous reagents. The adsorp-

todegradation was analyzed using a chromatographic system from

tion process was conducted under the optimal condition pH = 2

Dionex consisting of a P580 A LPG gradient pump, an ASI-100

and from high concentrated FePC solution (Tables S1 and S2). The

autosampler, an STH 585 oven and an RF 2000 ﬂuorescence detec-

acidic environment promotes the adsorption of iron phthalocya-

tor. Samples of exactly 5 ␮L were injected into a C18 Hypersil GOLD

nine/tetrasulfonic acid onto spongin, due to the interaction of the

column (150 mm × 4.6 mm I.D.; 5 ␮m) with a 2.1 mm I.D. ﬁlter car-

protonated amino groups of spongin with the sulfonic groups of

tridge (0.2 ␮m) from Thermo Scientiﬁc. The mobile phase consisted

the dye.

of solvent A (20% methanol) and solvent B (methanol) at a ﬂow rate

◦

of 1.5 mL/min at 35 C.

3.1. X-ray photoelectron spectroscopy (XPS)

2.3.3. HPLC-MS analysis

Identiﬁcation of the degradation products was performed using X-ray photoelectron spectroscopy was used to investigate the

HPLC-MS analysis. For this purpose the Ultimate 3000 HPLC system surface composition of the spongin and iron phthalocyanine and

from Dionex coupled with a QTRAP 4000 mass spectrometer from the obtained hybrid material SFE 400, and to determine the exact

ABSciex was used. Samples of exactly 10 ␮L were injected into a chemical states of the species on the surface of the materials. The

Gemini-NX C18 column (100 mm 2.0 mm I.D.; 3 ␮m) from Phe- XPS wide scan (Fig. 2) of the spongin identiﬁes carbon, nitrogen, and

◦

nomenex maintained at 35 C. The mobile phase consisted of 5 mM oxygen as the main constituents of the biopolymer and provides

ammonium acetate in water and methanol at ﬂow rate 0.3 mL/min valuable information about the chemical states of elements there.

on a gradient from 50% to 100% methanol in 2 min and then 4 min in Spongin has a still unknown chemical structure, but according to

isocratic conditions. The electrospray (ESI) ion source operated in previous studies [43,44] and observed binding energies, the pres-

negative mode. Nitrogen was used in both the source and the mass ence of C H ∼283 eV; C C, C C, C OH ∼285 eV; N C N, C O C

spectrometer. The following parameters of the ESI source and mass ∼286 eV and C O (N C O, O C O) ∼288 eV functional groups is

spectrometer were used: curtain gas pressure 10 psi, nebulization highly possible. Deconvoluted spectra of oxygen reveal the pres-

gas pressure 45 psi, auxiliary gas pressure 45 psi, source temper- ence of C O (531 eV) and C O (533 eV) groups. The XPS N 1s

◦

− −

ature 450 C, ESI voltage 4500 V, declustering potential 40 V. spectrum clearly indicates that peaks of nitrogen functionalities

m/z

Spectra were collected in scan mode in the range 70–500 . appear at 398 eV (the N in C N bonds), 399 eV (the N in NH2)

82 M. Norman et al. / Journal of Hazardous Materials 347 (2018) 78–88

surface of the FePC/biopolymer hybrid and changes in binding ener-

gies were observed, occurring especially at the sulfonated group

site.

3.2. EDS, elemental analysis and FTIR study

The results from energy dispersive spectroscopy and elemental

analysis are given in Table 1.

These results present the expected elemental contents for spon-

gin and iron phthalocyanine. Spongin consists mostly of carbon,

nitrogen, oxygen and hydrogen. The presence of sulfur is connected

with the disulﬁde bonds of cysteine, which has also been found in

spongin previously [18]. Some other trace elements revealed by

EDS may have been incorporated into the spongin structure from

the environment. Elemental and EDS analysis of the sample result-

ing from the adsorption of iron phthalocyanine on the surface of

the spongin showed average percentage values for the contents

Fig. 4. Thermogravimetric curves of spongin, iron phthalocyanine and a sample

of elements which are common to both initial materials. More-

obtained after adsorption from 400 mg/L FePC solution.

over, an increase in iron (0.52%) appears in the SFe 400 sample. The

analysis provides proof of the different elemental compositions of

and 401 eV (the N in C N bonds) (deconvoluted spectra are not the various samples and indirectly conﬁrms the effectiveness of

presented). preparation of the spongin skeleton/phthalocyanine material.

The XPS spectrum of FePC, apart from C 1s, N 1s and O 1s, The results of FTIR analysis are depicted in Fig. 1S, and

additionally reveals the presence of S 2p (167, 168, 170 eV – conﬁr- assignments of the band positions are presented in Table S4 (Sup-

2−

mation of SO4 groups), Na 2p (1070 eV) and Fe 2p (708 eV). After plementary material). Norman et al. have published a detailed

the adsorption process these elements are also observed on the SFe analysis of the FTIR spectrum of spongin from the H. communis

400 spectrum, but with the peak maxima slightly shifted. More- skeleton [41]. The assignments of vibrational frequencies charac-

over, the Fe 2p spectrum can be divided into two main peaks at 710 teristic for FePC were made according to Paul and Joseph [47] and

and 714 eV, which are assigned respectively to Fe 2p3/2 and Fe 2p1/2 Berrios et al. [48].

asymmetric bands [45]. The Fe 2p3/2 peak is due to the presence of

Fe(III) species [46].

No signiﬁcant changes were found in the bond energies of the 3.3. Carbon-13 nuclear magnetic resonance ( C CP MAS NMR)

aforementioned components following impregnation of the spon-

gin with iron phthalocyanine. Nevertheless, besides the elements The spectra from carbon nuclear magnetic resonance analysis

carbon, oxygen and nitrogen, iron and sulfur were detected on the of spongin, SFe 400 and SFe 500 are depicted in Fig. 3. The C CP

Fig. 5. Degradation efﬁciency of BPA, phenol, chlorophenol and ﬂuorophenol, as a function of time, under various experimental conditions. (For interpretation of the references

to colour in the text, the reader is referred to the web version of this article.)

M. Norman et al. / Journal of Hazardous Materials 347 (2018) 78–88 83

Table 2

Kinetic parameters of phenol, chlorophenol, ﬂuorophenol and BPA degradation.

Kinetic model phenol 20 mg/L ch-phenol f-phenol 20 mg/L BPA 20 mg/L BPA 50 mg/L

2 2 2 2 2

k r k r k r k r k r

0 0.1726 0.8457 0.0798 0.7836 0.2213 0.6155 0.4373 0.7072 0.2679 0.8997

I 0.0136 0.9291 0.0293 0.7887 0.0287 0.8359 0.0918 0.954 0.0661 0.8898

II 0.0011 0.9815 0.0052 0.9445 0.005 0.9826 0.0251 0.9793 0.0076 0.9977

MAS NMR spectrum of spongin has been described in our previous NMR chemical shifts with varying concentration of iron phthalo-

work [49]. cyanine.

The analysis reveals shifts in signal maxima and the formation

of new peaks in the range 10–80 ppm, which suggests chemical

3.4. Thermal analysis

interaction between the spongin and the adsorbed dye. The sig-

nal at 172.1 ppm (from carbonyl groups) in the hybrid material is

To show the major mass loss temperatures, the TG curves are

split and shifted to lower values (169.8 and 166.2 ppm for SFe 400

presented in Fig. 4.

and 169.3 and 165.2 ppm for SFe 500). This fact implies that these

The data obtained for spongin indicate that this material is ther-

bonds are involved in the formation of interactions between the ◦

mally stable up to 210 C. Above that temperature a considerable

two constituents. Moreover, there was a slight change in the C

mass loss (60%) occurs, associated with changes in the spongin

Fig. 6. Plots of Ct /C0 vs. time, and kinetics of photodegradation (inner plots).

84 M. Norman et al. / Journal of Hazardous Materials 347 (2018) 78–88

Fig. 7. Averaged mass spectrum taken from the chromatogram of degraded bisphenol A (sampling time-point 20 min). Each peak in the mass spectrum is described with the

m/z value of the deprotonated ion, the retention time of the respective chromatographic peak and the chemical structure of the molecule.

structure caused by melting of the ˛-chains of spongin (in analogy The results depicted as a blue line – measurements with the

to the process involving ˛-keratin) and breakdown of cross-links use of UV light, H2O2 and phthalocyanine spongin hybrid material

including S S bonds, hydrogen bonds and salt links, which stay together – demonstrate the effective functioning of SFe 400 as a cat-

in accordance with [18,19]. At higher temperatures a very large alyst. One hour of irradiation ensures 100% efﬁciency. Moreover, the

mass loss is observed, amounting to 70% of the sample mass, linked degradation is very fast: after 10 min of the process only 5% of the

to the rupture of peptide bonds. The thermal degradation of FePC BPA remains in the reactor. In the case of phenol and its derivatives,

occurs in several steps, involving the loss of peripheral substituents decomposition is faster for chlorophenol and ﬂuorophenol (30 min)

and destruction of a macroheterocycle [50]. than for phenol (50 min), but still 100% efﬁciency is reached. The

In the case of the hybrid product, after initial loss of moisture at positive results motivated us to extend the study and increase the

◦ ◦

80–120 C, a 55% mass loss occurred at 470 C. The hybrid material concentration of pollutants from 2 to 20 mg/L. In these conditions

exhibits greater thermal stability than pure spongin. the degradation efﬁciency for chloro-and ﬂuorophenol is similar

(85%), and that for phenol is 60%. The higher rate of degradation for

chloro- and ﬂuorophenol is explained by the fact that substitution

by chlorine or ﬂuorine decreases the probability of photodissocia-

3.5. Catalysis investigation

tion of the O H bond in phenol, because it decreases the population

efﬁciency of the photodissociative state [51]. For BPA, complete

In order to investigate the catalytic activity of the prepared

degradation took place even with a 50 mg/L solution.

heterogeneous catalysts, oxidation of bisphenol A (BPA), phenol,

The percentage degradation of the analyzed compounds is neg-

chlorophenol and ﬂuorophenol with hydrogen peroxide as a green

ligibly small with ultraviolet radiation only and also with hydrogen

oxidant with UV assistance was studied.

peroxide (without ultraviolet irradiation), but the synergistic effect

The results of the degradation experiment using the above-

of UV and H2O2 results in a marked enhancement of the rates of

mentioned phenolic compounds clearly indicate that the spongin

degradation. This may be explained by the fact that in an aqueous

with adsorbed iron phthalocyanine efﬁciently works as a catalyst −

solution, hydrogen peroxide dissociates to form HO2 anions and

in a degradation process enhanced by UV light and hydrogen per-

O2 in a chain reaction, while in the presence of UV light hydrogen

oxide. To verify if and how ultraviolet radiation and the external •

peroxide generates OH free radicals, which exhibit higher oxida-

oxidant (H2O2) catalyze the process, additional experiments were

tion potential [52]. On the other hand, excessive H2O2 molecules

carried out. From the data in Fig. 5, it is apparent that UV light (dark •

consume hydroxyl radicals (OH ) and form hydroperoxyl radicals

pink line) not nearly degrade phenol and BPA within 1 h of mea- •

(HO2 ). This radical is less reactive and does not contribute to

surement. For comparison, ultraviolet irradiation causes 35% and

the degradation [53]. The calculated synergy index of photooxi-

45% decomposition of chloro- and ﬂuorophenol respectively. The

dation for 2 mg/L was 2.15 for BPA, 2.46 for phenol, and 1.06 and

addition of hydrogen peroxide (cyan line) in fact decreases these

1.02 for chloro- and ﬂuorophenol respectively. The test of cata-

values to 25% and 20% in the same period of time, and for phe-

lyst’s reusability was also performed and the results are detailed

nol and BPA the efﬁciency does not change. The experimental data

described in Supplementary material – Table S3.

show that phenolic compounds are not adsorbed onto spongin with

In previous research modiﬁed oxides (CuOx, ZnO, TiO2) have

iron phthalocyanine (pink line) (no more than 10%, irrespective of

most often been used as catalysts for BPA degradation [53–57].

the compound), but the presence of UV light enhanced the process

In most reported experiments the removal efﬁciency was close

(dark cyan line).

M. Norman et al. / Journal of Hazardous Materials 347 (2018) 78–88 85

Fig. 8. Averaged mass spectrum taken from the chromatogram of degraded (A) phenol, (B) chlorophenol and (C) ﬂuorophenol (sampling time-point 10 min). Each peak in the

mass spectrum is described with the m/z value of the deprotonated ion, the retention time of the respective chromatographic peak and the chemical structure of a molecule.

to 100%, but the concentration of BPA was lower (10 mg/L), and the ﬁndings of other studies that UV light and the presence of

the process required longer irradiation (24 h) or a complicated hydrogen peroxide promote the degradation process [58]. Similar

procedure of catalyst preparation. Our results are consistent with observations have been reported for phenol, chlorophenol and ﬂu-

86 M. Norman et al. / Journal of Hazardous Materials 347 (2018) 78–88

orophenol degradation. Mecha et al. [35] demonstrated that the Moreover, UV irradiation also affects the metal phthalocyanine

doping of TiO2 with metals enhanced the degradation of these [35]:

compounds. It is also reported that iron(II) phthalocyanine sup- ∗ + −

FePC + hv → FePC → FePC + e

ported on graphene [35] or zeolites [59] functions efﬁciently as a

catalyst in the decomposition of phenol. Photocatalytic activity is

− • −

greatly enhanced because of the ␲–␲ stacking interaction when e + H2O2 → OH + OH

FePC is loaded on graphene, but the addition of the oxidant, H2O2,

Hydroxyl radicals lead to the rapid and non-selective oxidation

is essential.

of many organic compounds. The analysis of degradation products

•

conﬁrms the activity of OH .

3.5.1. Kinetic studies

Results for degradation kinetics were calculated according to

3.5.3. Identiﬁcation of degradation products

equations presented in Supplementary material, but data are

Degradation of BPA takes place according to previously

shown only for the best experimental conditions (SFe + UV + H2O2).

described schemes [53,57,64–66] combining oxidation of phenolic

Generally, independently of the removed compounds, for a less

ring and oxidative scission at the sp carbon atom interconnect-

concentrated solution (2 mg/L) of BPA, phenol, chlorophenol and

ing the two phenolic rings. The structures of a number of detected

fluorophenol the pseudo-first-order reaction model fitted the

degradation products are presented in Fig. 7.

experimental data more closely. However, the results for 20 mg/L

The averaged mass spectrum taken from the chromatogram

(and in the case of BPA, also for 50 mg/L) demonstrated a linear rela-

shows different intensities of ions reﬂecting the different quanti-

tionship with a slope equal to the pseudo-second-order reaction

ties of compounds present in the samples. The most abundant ion

rate constant. Values of the rate constant and correlation coef-

2 at m/z = 243.1 represents deprotonated BPA catechol, with abun-

ﬁcients (r ) are presented in Table 2. As can be seen from the

dance higher than that of deprotonated BPA at m/z = 227.0. Other

kinetic curves (Fig. 6) it is clear that the photodegradation of BPA

important degradation products include BPA 3,4-quinone with a

is most intense (as evidenced by the shape of the Ct /C0 vs. t curve).

deprotonated ion at m/z = 241.1, eluting with a similar retention

The data show that phenol is not fully degraded, but that chloro-

time to BPA catechol. The main compounds formed by scission

and ﬂuorophenol are much better degraded. Based on the Ct /C0

include 2-phenyl isopropanol (the ion at m/z = 151.0) and iso-

curve, it can be assumed that extending the degradation time would

propenyl catechol (the ion at m/z = 149.0). Ions of the other products

increase the efﬁciency of the process. In the case of the chloro- and

formed after scission were also found, but their abundances were

ﬂuoro- derivatives 60 min is the optimal time, as the system is then lower.

approaching equilibrium.

Degradation of phenol, ﬂuorophenol and chlorophenol takes

place according to two pathways. In the ﬁrst, oxidation of the ring

3.5.2. Mechanism

takes place, leading to catechols. Characteristic ions were found at

The photochemical process of degradation employing H2O2 and

m/z 16 daltons higher than for the parent phenols, as shown in Fig. 8.

UV belongs to a set of chemical treatment procedures applied in •

The other pathway involves OH attack with formation of a rad-

wastewater treatment and called AOP (Advanced Oxidation Pro-

ical, which then attacks the second molecule leading to dimers,

cesses) [60,61].

as has been previously reported for phenol [67]. The ions found

The degradation pathway is based on a photooxidation pro-

for degradation compounds formed by this pathway are present

cess. The most reactive species causing decomposition of organic

in the mass spectra from two experiments: from the degradation

compounds is the hydroxyl radical, which is generated in several

of chlorophenol (the ion at m/z = 253.0) and ﬂuorophenol (the ion

reactions:

at m/z = 221.0). Similar ions were not found in the degradation of

– photodissociation of hydrogen peroxide under UV irradiation:

phenol, which can be attributed to its lower ionization (similarly to

the lower abundance of deprotonated phenol than of deprotonated

•

H2O2 + hv → 2OH chlorophenol and ﬂuorophenol).

– in reaction with FePC:

4. Conclusions

III III

–Fe + H2O2 → –Fe H2O2

In summary, the preparation and properties of the designed

III II + photocatalyst SFe have been investigated in detail for the ﬁrst

–Fe H2O2 → –Fe + HO2 + H

time. The adsorption efﬁciency is strongly dependent on the pH.

The produced material was thoroughly investigated and results

II III − •

+ → + +

–Fe H2O2 –Fe OH OH of physicochemical analysis conﬁrmed the successful deposition

of phthalocyanine. For example FTIR spectroscopy show a charac-

Hydroxyl radicals are generated by the decomposition of H2O2 −1

teristic signal at 1026 cm is a clear indication of the presence

on the surface of the solid catalyst through a surface complexion

of SO3H, originated from phthalocyanine, in the SFe 400 hybrid.

mechanism [62]. In a publication of Rodriquez et al. these reactions

Iron phthalocyanine immobilized on spongin scaffold is a suit-

are presented as follows [63]:

able solid photocatalyst for the degradation of organic compounds.

+ + + •

3 2 Hydroxyl radicals, ultraviolet light, an adsorption process and

Fe + H2O2 → Fe + H + HO2

the metal phthalocyanine’s photosensitizing properties in com-

3+ • 2+ + bination are responsible for degradation, indicating a synergistic

Fe + HO2 → Fe + H + O2

effect. Moreover, degradation efﬁciency increases with time but

decreases with concentration for phenol, chloro- and ﬂuorophenol,

• •

+ → + +

HO2 H2O2 H2O O2 OH for BPA the removal efﬁciency is 100%, regardless of concentration.

The degradation process, for 20 and 50 mg/L solutions, followed

– in reaction with FePC and UV irradiation [35]:

a pseudo-second-order kinetic model. Several intermediate com-

+ + • +

3 2 pounds were identiﬁed, making it possible to propose possible

Fe + H2O2 + hv → Fe + OH + H

pathways for BPA and phenol degradation. BPA undergoes a series

M. Norman et al. / Journal of Hazardous Materials 347 (2018) 78–88 87

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Ortiz, D.D. Dionysiou, Magnetically recoverable TiO2-WO3 photocatalyst to Prof. Dr. rer. nat. habil Hermann Ehrlich Freiberg 11.12.2017 Institute of Experimental Physics TU Bergakademie Freiberg Leipziger Str. 23 09599 Freiberg Germany

Statement of the co-authorship

I confirm particiaption in the following publications:

1. Małgorzata Norman, Przemysław Bartczak, Jakub Zdarta, Włodzimierz Tylus, Tomasz Szatkowski, Allison L. Stelling, Hermann Ehrlich, Teofil Jesionowski, Adsorption of C.I. Natural Red 4 onto spongin skeleton of marine demosponge, Materials 2014, 8, 196-216

2. Małgorzata Norman, Przemysław Bartczak, Jakub Zdarta, Hermann Ehrlich, Teofil Jesionowski, Anthocyanin dye conjugated with Hippospongia communis marine demosponge skeleton and its antiradical activity, Dyes and Pigments 2016, 134, 541- 552

3. Małgorzata Norman, Przemysław Bartczak, Jakub Zdarta, Wiktor Tomala, Barbara Żurańska, Anna Dobrowolska, Adam Piasecki, Katarzyna Czaczyk, Hermann Ehrlich, Teofil Jesionowski, Sodium copper chlorophyllin immobilization onto Hippospongia communis marine demosponge skeleton and its antibacterial activity, International Journal of Molecular Sciences 2016,17(9), 1564-1580

4. Małgorzata Norman, Jakub Zdarta, Przemysław Bartczak, Adam Piasecki, Iaroslav Petrenko, Hermann Ehrlich, Teofil Jesionowski, Marine sponge skeleton photosensitized by copper phthalocyanine: A catalyst for Rhodamine B degradation, Open Chemistry 2016, 14, 243-254

5. Małgorzata Norman, Sonia Żółtowska-Aksamitowska, Agnieszka Zgoła-Grześkowiak, Hermann Ehrlich, Teofil Jesionowski, Iron(III) phthalocyanine supported on a spongin scaffold as anadvanced photocatalyst in a highly efficient removal process ofhalophenols and bisphenol, Journal of Hazardous Materials 2018, 347, 77-88

My participation in each publications is estimated at 10% and includes planning the experiment and developing of the results.

mgr in. Przemysław Bartczak Poznań 30.08.2017 Wydział Technologii Chemicznej Politechnika Poznańska ul. Berdychowo 4 60-965 Poznań

Statement of the co-authorship

I confirm particiaption in the following publications:

3. Małgorzata Norman, Przemysław Bartczak, Jakub Zdarta, Wiktor Tomala, Barbara urańska, Anna Dobrowolska, Adam Piasecki, Katarzyna Czaczyk, Hermann Ehrlich, Teofil Jesionowski, Sodium copper chlorophyllin immobilization onto Hippospongia communis marine demosponge skeleton and its antibacterial activity, International Journal of Molecular Sciences 2016,17(9), 1564-1580

My participation in each publications is estimated at 5% and includes calculation of the adsorption kinetics and isotherms parameters.

dr Allison L. Stelling Durham 30.08.2017 Department of Biochemistry Duke University Medical Center 307 Research Drive, 228B Durham NC 27710 USA

Statement of the co-authorship

I confirm particiaption in the following publication:

1.! Małgorzata Norman, Przemysław Bartczak, Jakub Zdarta, Włodzimierz Tylus, Tomasz Szatkowski, Allison L. Stelling, Hermann Ehrlich, Teofil Jesionowski, Adsorption of C.I. Natural Red 4 onto spongin skeleton of marine demosponge, Materials 2014, 8, 196-216

My participation is estimated at 5% and include language proofs of the whole manuscript.