FLAVIA CALLEFO
A UTILIZAÇÃO DE BIOMINERAIS COMO POSSÍVEIS BIOASSINATURAS MICROBIANAS
CAMPINAS 2018
A UTILIZAÇÃO DE BIOMINERAIS COMO POSSÍVEIS BIOASSINATURAS MICROBIANAS
TESE APRESENTADA AO INSTITUTO DE GEOCIÊNCIAS DA UNIVERSIDADE ESTADUAL DE CAMPINAS PARA OBTENÇÃO DO TÍTULO DE DOUTORA EM CIÊNCIAS NA ÁREA DE GEOLOGIA E RECURSOS NATURAIS.
ORIENTADORA: PROFª DRA. FRESIA SOLEDAD RICARDI TORRES BRANCO
COORIENTADOR: PROF. DR. FABIO RODRIGUES
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO/TESE DEFENDIDA PELA ALUNA FLÁVIA CALLEFO E ORIENTADA PELA PROFª. DRA. FRESIA SOLEDAD RICARDI TORRES BRANCO.
CAMPINAS 2018
Ficha catalográfica
Universidade Estadual de Campinas Biblioteca do Instituto de Geociências
Marta dos Santos - CRB 8/5892
Callefo, Flavia,
1983-C132u CalA utilização de biominerais como possíveis bioassinaturas microbianas / Flavia Callefo. – Campinas, SP : [s.n.], 2018.
CalOrientador: Fresia Soledad Ricardi Torres Branco.
CalCoorientador: Fabio Rodrigues.
CalTese (doutorado) – Universidade Estadual de Campinas, Instituto de Geociências.
Cal1. Magnetita. 2. Biofilme. 3. Paleomagnetismo. 4. Síncrotron. 5. Microrganismos. I. Ricardi-Branco, Fresia, 1963-. II. Rodrigues, Fabio. III. Universidade Estadual de Campinas. Instituto de Geociências. IV. Título.
Informações para Biblioteca Digital
Título em outro idioma: The use of biominerals as possible microbial biosignatures Palavras-chave em inglês: Magnetite Biofilm Paleomagnetism Synchrotron Microorganisms
Área de concentração: Geologia e Recursos Naturais Titulação: Doutora em Geociências
Banca examinadora:
Fresia Soledad Ricardi Torres Branco [Orientador] Carolina Zabini
Gelvam André Hartmann Marcelo Adorna Fernandes
Miriam Liza Alves Forancelli Pacheco
Data de defesa: 20-04-2018
Programa de Pós-Graduação: Geociências
AUTORA: Flavia Callefo
A UTILIZAÇÃO DE BIOMINERAIS COMO POSSÍVEIS BIOASSINATURAS MICROBIANAS
ORIENTADORA: Profa. Dra. Fresia Soledad Ricardi Torres Branco COORIENTADOR: Prof. Dr. Fabio Rodrigues
Aprovado em: 20 / 04 / 2018
EXAMINADORES:
Profa. Dra. Fresia Soledad Ricardi Torres Branco - Presidente
Prof. Dr. Gelvam André Hartmann
Prof. Dr. Marcelo Adorna Fernandes
Profa. Dra. Carolina Zabini
Profa. Dra. Mirian Liza Alves Forancelli Pacheco
A Ata de Defesa assinada pelos membros da Comissão Examinadora, consta no processo de vida acadêmica do aluno.
Dedico este trabalho a todas as mulheres cientistas, especialmente paleontólogas, que lutam diariamente pela igualdade que já deveria existir.
Este trabalho jamais poderia ter sido realizado se não fosse os esforços de minha querida orientadora Profa. Dra. Fresia Ricardi-Branco, a quem sempre me deu todo o suporte necessário, sempre respeitou meu trabalho e autonomia, me encorajando e dando o exemplo para seguir em frente apesar de toda e qualquer adversidade.
Agradeço às instituições e empresa que viabilizaram o desenvolvimento da pesquisa: Instituto de Geociências da Unicamp; Laboratório Nacional de Luz Sincrotron e Laboratório Nacional de Nanotecnologia; Old Dominion University e Carnegie Institution of Science, EUA; Laboratório de Paleomagnetismo e Magnetismo de Rochas do Instituto de Astronomia, Geofísica e Ciências Atmosféricas da USP; Universidade de Brasília e Votorantim Metais S/A.
Aos órgãos de fomento: CAPES, pela bolsa de doutorado concedida e pelo financiamento do período nos Estados Unidos; FAPESP pelo financiamento do projeto que viabilizou os campos e a compra de materiais e infraestrutura necessários.
À Dra. Nora Noffke, da Old Dominion University, EUA, pelo auxílio nas pesquisas, escrita dos artigos e amizade durante o período fora do país, fazendo-me sentir em casa.
À Frances, Silver e Bethsy Ibenye, por terem sido minha família nos Estados Unidos.
A todos os meus colaboradores (citados nos artigos).
Ao meu co-orientador Dr. Fabio Rodrigues por todo o apoio oferecido.
Ao Dr. Douglas Galante, pelo incentivo, participação ativa nesta pesquisa e por sempre me encorajar a sonhar alto.
Aos colegas/amigos Lara Maldanis e Gabriel Ladeira Osés, pelo auxílio nas análises no LNLS.
Aos professores do Instituto de Geociências, especialmente Dra. Sueli Yoshinaga, Dra. Jacinta Enzweiler, Dr. Gelvam Hartmann, Dr. Emilson Pereira Leite e Dra. Carolina Zabini. Aos funcionários Cristiano, Érica, Gorete, Valdirene, Valdir e Cristina, por sempre colaborarem para tudo funcionar corretamente.
Aos amigos Érica Rodrigues, Ana Érika, Francisco, Isabel, Amanda Midori, Mel, Juliana Sampaio, Sandra Tavares, Deborah Horta, Ariel e Bia, Daiane Belgini, Artur Camara, Diego Silva, Nanci, Ju e Leo, Rogério Salustiano, Emilson, Kelly e Amanda.
À minha prima Michele Leardini, pela inspiração, amizade e incentivo fundamentais.
Aos meus tios e primos (muito numerosos para listar).
Aos meus pais (Ana e Valdomiro), minha avó Helena, sogros (Gláucia e Dionísio), irmãs (Fernanda e Anelize) e sobrinho Felipe, que sempre estiveram ao meu lado e me encorajaram a seguir meus sonhos.
E finalmente à minha família, Rafael Amaral Cataldo, com seu suporte emocional, paciência, encorajamento e palavras de incentivo que nunca me deixaram sentir que eu deveria desistir; e Predador, meu cachorro/filho, que mesmo sem dizer uma palavra, me deixa feliz só pelo fato de existir.
Flávia Callefo
Licenciada em Ciências Biológicas pelo Instituto de Biologia da Universidade Estadual de Campinas (2011), Mestre em Geociências pelo Instituto de Geociências da Universidade Estadual de Campinas (2014) e Doutora em Geociências, Área de Geologia e Recursos Naturais, pela Universidade Estadual de Campinas (2018).
Possui experiência em Paleontologia, especialmente no estudo de estruturas microbianas (microbialitos, esteiras microbianas e MISS - microbially induced sedimentary structures) modernas e fósseis. A ênfase de seu trabalho consiste em detecção de bioassinaturas geoquímicas e morfológicas e interpretações paleoambientais. Também atua em tema relacionado à influência de metazoários na construção de estruturas microbianas.
Atualmente, colabora em pesquisas relacionadas à detecção de biominerais e é membro do Subcomitê de Estratigrafia do Pré-Cambriano (Subcommission on Precambrian Stratigraphy) do ICS (International Commission on Stratigraphy).
Desde o seu surgimento e evolução no Arqueano, os microrganismos têm o potencial de interagir e participar do ciclo dos elementos na Terra. A evolução da capacidade de se organizar em biofilmes garantiu a sobrevivência de suas espécies ao longo do tempo geológico, conferindo uma série de benefícios às comunidades microbianas, como proteção e máximo aproveitamento do microambiente colonizado. Outra vantagem adaptativa importante para seu sucesso foi a habilidade de realizar a biomineralização, que dentre diversas vantagens para a sobrevivência das espécies envolvidas, também possibilitou o surgimento de uma maior diversidade mineral na crosta terrestre. Apesar da dificuldade de detecção, os biominerais possuem algumas características cristalográficas e magnéticas que podem contribuir para diferenciá-los dos minerais formados inorganicamente. A soma destas características detectáveis com bioassinaturas morfológicas, como a rara presença de biofilmes e/ou de fósseis de microrganismos, pode atestar para a origem biogênica destes minerais. Esta pesquisa teve como objetivo principal a avaliação do potencial dos biominerais como bioassinaturas em rochas de contextos geológicos e idades diferentes, com a aplicação de diversas técnicas convencionais de bancada (petrografia, MEV/EDS, TEM e Espectroscopia Raman), técnicas de luz síncrotron (µ-XRF, XANES e XRD) e técnicas magnéticas (curvas de histerese, FORC, IRM e curvas de aquisição de magnetização em baixas temperaturas). Com o conjunto de dados obtidos, foi possível utilizar biominerais para a diagnose de biogenicidade de estruturas sedimentares outrora consideradas de origem inorgânica; avaliar a preservação de bioassinaturas geoquímicas em estromatólitos proterozóicos e utilizá-los para tecer interpretações paleoambientais. Informações como a periodicidade de deposição de sedimentos, influência de temperatura e de processos diagenéticos, como o hidrotermalismo, e principalmente a influência biológica nos contextos deposicionais foram possibilitadas através da interpretação dos resultados obtidos. Sendo assim, este estudo demonstrou o potencial dos biominerais como bioassinaturas microbianas no registro geológico.
Since its appearance and evolution during the Archean, microorganisms have the potential to interact and participate of the cycles of elements on Earth. The evolution of the capability to organize themselves in biofilms allowed their evolutive success. The benefits to the microbial community are numerous, such as protection and maximum exploitation of the colonized microenvironment. Another important evolutionary step for its success was the ability to perform biomineralization, which offered several advantages for the survival of the species involved, also allowing the emergence of a mineral diversity on the Earth's crust. The biominerals present crystallographic and magnetic characteristics that may help to differentiate them from inorganic minerals. These characteristics plus the morphological biosignatures, such as the rare presence of fossilized microorganisms and/or biofilms, may be a proof for the bionegic origin of these minerals. This research had as main objective the application of several conventional techniques (petrography, SEM/EDS, TEM and Raman Spectroscopy), synchrotron-based techniques (µ-XRF, XANES e XRD) and magnetic techniques (hysteresis loop, FORC, IRM and low temperature magnetization measurements) in order to detect biominerals as biosignatures in rocks from different depositional and geological contexts. The specific objectives were the utilization of these biominerals to diagnose the biogenicity of Permian sedimentary structures and to evaluate the preservation of geochemical biosignatures in proterozoic stromatolites. These biosignatures were fundamental to obtain information regarding the paleoenvironment from the different sites of study. Information about the periodicity of the deposition of sediments, the influence of temperature and the diagenetic processes, such as hydrothermalism, was acquired through the interpretation of the obtained results.
1.1 Biomineralização ... 15
1.2 Biominerais ... 16
2. JUSTIFICATIVAS ... 19
3. OBJETIVOS ... 20
4. ARTIGOS ELABORADOS ... 21
4.1 Artigo submetido: “Microbially Induced Sedimentary Structures (MISS) in Rhythmites of Itararé Group (Brazil) and their Paleoenvironmental Implications” .... 22
Abstract ... 23
Introduction ... 24
Study area and geological setting ... 25
Material and methods ... 27
Results and interpretations ... 31
Discussion ... 41
CONCLUSIONS ... 47
ACKNOWLEDGMENTS... 48
REFERENCES ... 49
4.2 Artigo a submeter: “Fossilized biofilms in Meso-Neoproterozoic stromatolites from Vazante Group, Brazil” ... 54
ABSTRACT ... 55
GEOLOGICAL SETTING ... 57
MATERIAL AND METHODS ... 58
RESULTS ... 61
DISCUSSIONS ... 72
5. CONCLUSÕES ... 87
6. REFERÊNCIAS... 90
ANEXO: Artigo submetido “Microbial biofacies in Holocene deposits of the Lagoa Salgada, Rio de Janeiro State, Brazil”. ... 93
1. INTRODUÇÃO
Desde o seu surgimento na Terra e, principalmente após alcançarem maior complexidade metabólica, os microrganismos são capazes de interagir nos processos de formação e evolução da crosta terrestre, exercendo um papel importante nos ciclos dos elementos, formação e evolução mineral, e na composição da atmosfera (Des Marais 2000; Hazen et al. 2008). Seu sucesso evolutivo se deu, dentre outros fatores, pela capacidade de se organizarem em comunidades denominadas biofilmes, nas quais as colônias microbianas ficam envoltas pela substância polimérica extracelular (EPS) secretada pelas próprias células bacterianas (Costerton et al. 1995; Stoodley et al. 2002; Decho et al. 2000). Protegidos pelo biofilme, os microrganismos criam um microambiente específico e ideal para a proteção das colônias contra estresses ambientais, realização de trocas de metabólitos, proliferação das células, dentre outros benefícios. Quando diferentes comunidades bacterianas que compõem os biofilmes se organizam e ganham maior escala de desenvolvimento, são formadas as esteiras microbianas (Neu 1994; Davey & O´Toole 2000; Noffke & Arwramik 2013). Estas podem se desenvolver a ponto de haver estratificações metabólicas de acordo com as necessidades de luz, oxigênio, nutrientes e outros fatores (Figura 1). Por causa destas habilidades, as esteiras microbianas são consideradas os mais antigos ecossistemas da Terra (Noffke et al. 2006; Schopf 2006), e sua capacidade de criar o próprio substrato geológico e biológico permitiu a adaptação destes sistemas a uma extensa variedade de condições e estresses ambientais. Neste microambiente é possível ocorrer desde a reciclagem de metabólitos e até o reaproveitamento de material genético pelos microrganismos. Estas habilidades possibilitaram a prevalência das esteiras microbianas ao longo de toda história da Terra, desde seu surgimento no Arqueano (Dupraz et al. 2009).
Figura 1. Esteira microbiana moderna (Lagoa Vermelha, Rio de Janeiro), com estratificação hipotética de tipos metabólicos. (foto: Flávia Callefo, nov. 2016).
Quando ocorre a interação entre os microrganismos de um biofilme ou esteira microbiana com a dinâmica física dos sedimentos ainda não consolidados, os microrganismos podem criar estruturas e morfologias específicas denominadas MISS - microbially induced
sedimentary structures (Noffke et al. 1996; Gerdes et al. 2000, Noffke et al. 2001; Noffke 2010), que são estruturas melhor observadas no plano horizontal, na superfície de camadas sedimentares (Noffke & Arwramik 2013). Quando ocorre a precipitação de minerais, como por exemplo os de carbonato de cálcio (como calcita ou dolomita), induzida pelo metabolismo bacteriano, são formadas estruturas organo-sedimentares de formatos variados denominadas microbialitos, dentre elas os estromatólitos (Burne & Moore 1987; Riding 2000; Riding 2011; Noffke & Arwramik 2013). Ao contrário de MISS, os microbialitos são melhor caracterizados no plano vertical, no qual é possível apreciar suas características laminações repetitivas ao longo da estrutura.
A influência que os microrganismos exercem na evolução do planeta está relacionada com diferentes tipos de espécies e diferentes vias metabólicas específicas (Konhauser & Riding 2012). Com relação à atmosfera, essa influência pode ser exemplificada por meio das cianobactérias, que foram capazes de modificar a condição redox através da “inauguração” dos processos de fotossíntese (Kasting 1991; Des Marais 1995 e 2000; Kasting & Howard 2006). Com relação à crosta e os minerais e rochas que a compõe, os exemplos provêm da
interação micróbio/mineral, conhecida por meio de uma grande variedade de microrganismos como os quimilitotróficos (que ao obterem energia através da transferência de elétrons entre o meio e suas superfícies celulares, são capazes de gerar a precipitação de minerais ou reciclagem de elementos presentes na natureza). Independente da via metabólica empregada por cada tipo de microrganismo, os processos biogeoquímicos influenciam diretamente a química e a distribuição dos elementos no meio circundante das células. Nos processos de biomineralização, os processos químicos relacionados ao metabolismo bacteriano afetam o estado redox e de saturação dos fluidos ao redor das células. Os produtos metabólicos, como os minerais que podem ser precipitados, variam de acordo com a composição desses fluidos e do tipo de via metabólica empregada. Como consequência disto, uma ampla gama de biominerais microbianos é encontrada nos ambientes sedimentares (Konhauser 2007).
O termo "biomineralização" está relacionado aos processos pelos quais os organismos como bactérias, protozoários, fungos, plantas e animais formam minerais (Bazylinski 2001; Dove
et al. 2003). Grande parte dos biominerais produzidos é composta por carbonatos de cálcio,
silicatos e óxidos ou sulfetos de ferro (Bazylinski 2001).
1.1 Biomineralização
A capacidade de biomineralização foi uma importante garantia de sobrevivência dos microrganismos, que significou não só a garantia de sobrevivência de suas espécies, mas também revolucionou a geoquímica da Terra. O ciclo de diversos elementos como C, Ca, Mn, Mg, Fe, P, Si, S, entre outros, é fortemente afetado pelos processos de biomineralização (Konhauser & Riding 2012). Muitos microrganismos são capazes de formar fases minerais através de dois tipos de mecanismos (Konhauser & Riding 2012):
(i) a mineralização biologicamente induzida (Lowenstam 1981), na qual o mineral é produto de um processo passivo através da interação entre célula bacteriana e o ambiente no qual está inserida (como por exemplo, a dolomita ou calcita, que formam os estromatólitos). Nestes processos extracelulares, a química relacionada ao metabolismo bacteriano pode afetar o estado redox e de saturação dos fluidos ao redor das células. Os produtos metabólicos podem ser precipitados e variam de acordo com a composição desses fluidos e do tipo de via metabólica empregada. Como consequência disto, uma ampla gama de biominerais
microbianos é encontrada nos ambientes sedimentares e aquosos (Konhauser 2007);
(ii) a mineralização biologicamente controlada, na qual a mineralização é rigorosamente controlada pela genética dos organismos e resulta em biominerais que possuem um papel fisiológico ou estrutural para a célula (como por exemplo, as conchas de moluscos e a magnetita (ou greigita) que compõe os magnetossomos das bactérias magnetotáticas, um dos poucos procariontes envolvidos neste tipo de mineralização). Nestes processos intracelulares, o controle da precipitação do mineral é intrínseco, ou seja, atividades celulares e enzimáticas controlam a nucleação, crescimento, morfologia e alocação dos cristais (Dupraz et al. 2009).
1.2 Biominerais
O termo “biomineral” tem uma ampla gama de definições, e na maioria deles são referidos como minerais que foram produzidos por organismos vivos, possuindo componentes orgânicos e minerais (Weigner & Dove 2003; Skinner & Jahren 2003; Skinner 2005). De acordo com Dupraz et al. (2009), biominerais são produtos de uma mineralização biologicamente controlada, tendo uma participação intrínseca com relação à genética dos organismos, isto é, os minerais produzidos resultam em endo e exoesqueletos, por exemplo. Sendo assim, eles podem ser utilizados como evidências diretas de vida, assim como os somatofósseis. Em contraponto, os minerais que são produzidos de maneira induzida pela atividade metabólica de microrganismos, como o carbonato de cálcio que dá origem aos microbialitos, são considerados pelo autor como “organominerais”. Nesta tese, porém, o termo biomineral foi empregado no sentido mais amplo, como utilizado pelos autores Weigner & Dove (2003), Skinner & Jahren (2003) e Skinner (2005), definido anteriormente. Os biominerais possuem algumas propriedades específicas que podem ser utilizadas como parâmetros em sua diferenciação com relação aos minerais produzidos inorganicamente. Alguns desses parâmetros são: forma, cristalinidade, tamanho dos cristais, composição de elementos traços e isotópicos (Weiner & Dove 2003). A vantagem desta diferenciação é a possibilidade de detecção da origem desses minerais na natureza, ou seja, se foram precipitados por processos orgânicos ou inorgânicos. Para os minerais compostos por elementos que podem ser fracionados isotopicamente, a aplicação de técnicas isotópicas é a chave desta detecção.
Um estudo realizado por Thomas-Keprta et al. (2000) com magnetitas biogênicas (no caso, magnetossomos de bactérias magnetotáticas) e inorgânicas mostrou que ao menos seis características específicas os distinguem entre si, de modo a conferir vantagens aos organismos que as precipitam (como o aumento da eficiência da magnetização nos processos biologicamente controlados). Estas seis características são:
1) tamanho pequeno (domínio-simples, SD) dos cristais da magnetita biogênica, que permite que a partícula seja magnetizada uniformemente e não sofra consequências da variação de temperatura do ambiente na magnetização, ao contrário dos cristais maiores (multi-domínios, MD) apresentados pelas magnetitas inorgânicas;
2) pureza química conferida pelas magnetitas biogênicas, as quais constituem de cristalitos formados por Fe3O4 estequiométrico isentos de incorporações de outros elementos disponíveis no meio, como Al, Ti e Mn, como ocorre nas magnetitas precipitadas inorganicamente. Impurezas químicas reduzem a magnetização de saturação da magnetita;
3) perfeição cristalográfica (isenção de defeitos cristalográficos) das magnetitas biogênicas em relação às inorgânicas, afim de aumentar o momento magnético da partícula (magnetossomo). Esta característica é visível com microscopia eletrônica de transmissão (TEM);
4) organização em cadeia dos magnetossomos, com a finalidade de potencializar o momento magnético total da célula. Esta organização raramente é preservada em rochas antigas e fósseis, mas quando ocorre é um sinal de biogenicidade em relação a cristais únicos;
5) algumas formas de cristal de magnetita produzidas por algumas bactérias magnetotáticas são partículas isotrópicas, com morfologia incomum do cristal (por exemplo, em forma de agulha, alongadas e partículas prismáticas hexagonais) e não são típicas daquelas formadas inorganicamente, que geralmente exibem morfologias isotrópicas (por exemplo, cúbico, octaédrico, dodecaédrico);
6) tendência dos cristais de magnetita em magnetossomos ao alongamento ao longo do comprimento da corrente em uma das direções cristalográficas [111], não observável em magnetitas produzidas inorganicamente.
Em adição a estas características apresentadas, Abrajevitch et al. (2016) constatou que ao invés de apresentar grãos de tamanho superparamagnético (incapazes de apresentar uma carga de remanência), a massa biomineralizada estudada continha uma mistura de partículas de magnetita domínio-simples (SD) para pseudo-domínio simples (PDS) e magnetita multi-domínio (MD), que são capazes de transportar uma magnetização remanente estável.
2. JUSTIFICATIVAS
Cada vez mais se torna conhecido que a precipitação mineral na crosta terrestre tem uma participação orgânica maior do que se pensava outrora. A detecção de biominerais em rochas antigas da Terra, bem como o conhecimento dos processos envolvidos em sua formação e o funcionamento de biofilmes antigos e modernos, pode ser uma ferramenta muito útil para a elucidação do surgimento e evolução da vida na Terra. Este conhecimento também é importante para o maior entendimento do ciclo dos minerais no planeta, e além do mais, pode auxiliar na busca por vida fora da Terra. Com o planejamento de missões a Marte e com as pesquisas em satélites naturais e em outros exoplanetas, o entendimento das vias de formação de biominerais em sistemas terrestres precisam ser melhor entendidos (Bower et
al. 2015), assim como o desenvolvimento de métodos de detecção e, principalmente, a
diferenciação desses biominerais com relação aos minerais precipitados inorganicamente, o que ainda constitui um desafio para os pesquisadores.
3. OBJETIVOS
Esta pesquisa teve como objetivo central a investigação de biominerais como bioassinaturas microbianas em um conjunto de amostras de idades, contextos geológicos e deposicionais diferentes, com a utilização de multi-técnicas para a detecção destas bioassinaturas. Os objetivos específicos foram:
1) utilizar essas bioassinaturas para a diagnose de biogenicidade em rochas sedimentares classicamente consideradas produto de processos inorgânicos (artigo “Microbially Induced
Sedimentary Structures (MISS) in Rhythmites of Itararé Group (Brazil) and their Paleoenvironmental Implications”);
2) avaliar a preservação de bioassinaturas geoquímicas em rochas muito antigas (Meso-Neoproterozóico) para auxílio em interpretações paleoambientais, bem como a descrição da colonização de diferentes biofilmes em estruturas estromatolíticas (artigo: “Biofilmes
fossilizados em estromatólitos meso-neoproterozóico do grupo Vazante, Brasil”).
As amostras de (1) possuem idade Permiano-Carbonífera e pertencem à Bacia do Paraná, enquanto que as amostras de (2) possuem idade Pré-Cambriana e fazem parte do Grupo Vazante, Cráton São Francisco.
Além disso, foram estudados biofilmes microbianos atuais com o intuito de caracterizar a distribuição de biofácies, bem como a influência de metazoários em seu desenvolvimento. Foi submetido um artigo intitulado “Microbial biofacies in holocene deposits of the Lagoa
4. ARTIGOS ELABORADOS
“MICROBIALLY INDUCED SEDIMENTARY STRUCTURE (MISS) IN
RHYTHMITES OF THE ITARARÉ GROUP (CARBONIFEROUS/PERMIAN, BRAZIL) AND THEIR PALEOENVIRONMENTAL IMPLICATIONS”
Callefo, F., Ricardi-Branco, F., Hartmann, G.A., Galante, D., Rodrigues, F., Maldanis, L., Yokoyama, E., Teixeira, V. C., Noffke, N., Bower, D.M., Bullock, E.S., Coaquira, J.A.H, Fernandes, M.A.
“FOSSILIZED BIOFILMS IN MESO-NEOPROTEROZOIC STROMATOLITES FROM VAZANTE GROUP, BRAZIL”
Callefo, F., Ricardi-Branco, F., Noffke, N., Galante, D., Teixeira, V., Maldanis, L., Rodrigues, F., Bullock, E., Bower, D., Silva, A.M.
Anexo: “MICROBIAL BIOFACIES IN HOLOCENE DEPOSITS OF THE LAGOA SALGADA, RIO DE JANEIRO STATE, BRAZIL”
Ricardi–Branco, F., Callefo, F., Cataldo, R.F., Noffke, N, Pessenda, L.C.R., Vidal, A.C., Branco, F.C.
4.1 Artigo submetido: “Microbially Induced Sedimentary Structures (MISS) in Rhythmites of Itararé Group (Brazil) and their Paleoenvironmental Implications”
MICROBIALLY INDUCED SEDIMENTARY STRUCTURE (MISS) IN
RHYTHMITES OF THE ITARARÉ GROUP (CARBONIFEROUS/PERMIAN, BRAZIL) AND THEIR PALEOENVIRONMENTAL IMPLICATIONS
F. Callefoa*, F. Ricardi-Brancob, G.A. Hartmannb, D. Galantec, F. Rodriguesd, L. Maldanisc, E. Yokoyamae, V.C. Teixeirac, N. Noffkef, D.M. Bowerg,h, E.S. Bullocki, J.A.H. Coaquiraj, M.A. Fernandesk
a
Post-Graduation Program in Geosciences, Instituto de Geociências, Universidade Estadual de Campinas (UNICAMP), 13083-855, Campinas, Brazil;
b
Instituto de Geociências, Universidade Estadual de Campinas (UNICAMP), 13083-855, Campinas, Brazil;
c
Brazilian Synchrotron Light Laboratory (LNLS), Brazilian Center for Research in Energy and Materials (CNPEM), 13083-970, Campinas, Brazil;
d
Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, 05508-000, São Paulo, Brazil;
e
Instituto de Geociências, Universidade de Brasília, 70910-900, Brasília, Brazil f
Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, Norfolk, 23529, VA, USA;
g
Department of Astronomy, University of Maryland, College Park, MD, 20742, USA h
NASA Goddard Space Flight Center, Greenbelt, MD, 20771, USA i
Geophysical Laboratory, Carnegie Institution of Washington, 5251 Broad Branch Road, NW, Washington, D.C. 20015, USA.
j
Instituto de Física-Universidade de Brasília, 70919-970, Brasília, Brazil k
Laboratório de Paleoecologia e Paleoicnologia, Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Carlos., 13565-905, São Carlos, Brazil.
*Corresponding author.
Abstract
Microorganisms play a significant role in mineral precipitation. In this work is described the activity of microorganisms in sedimentary structures and mineral formation during the glacial period of the Gondwana supercontinent recorded by the “Itu rhythmites”. The Itu rhythmites are considered to be varve-type deposits that present alternating dark laminae (clay/silt-size sediments) and light layers (sand/gravel-size sediments) with varied thickness, forming couplets. The rock succession is of Permian to Carboniferous age and is located in the Paraná Basin, Brazil. Earlier studies focused on abiotic processes of structure formation and mineral precipitation. In this contribution, it was used different field and laboratory analyses to describe microbially-induced sedimentary structures (MISS) and biominerals such as magnetite and anatase. At the top of the stratigraphic succession of the Itu rhythmites, wrinkle structures, levelled ripple marks, and sinoidal desiccation cracks are interpreted as fossil microbial mats. Vertical sections show sinoidal structures and gas domes. All these macroscopic structures are interpreted as in-situ preserved microbial mats. Petrological, geochemical and magnetic analyses on the samples reveal a great variety of microscopic textures such as mat fabrics, oriented grains, and micro sequences. These together with geochemical signals support the interpretation as microbial structures. Therefore, we offer a new depositional model that considers the participation of microorganisms in the formation of laminae. Considering the effects of temperature and other factors in the bacterial productivity, the deposition of the last couplets of the outcrop has occurred in different seasons and different depositional processes, corroborating with the non-periodicity of 1 year per lithological couplet.
Introduction
During the Pennsylvanian-Cisuralian time, the Gondwana supercontinent experienced an ice age, the Late Paleozoic Ice Age (LPIA). During this time, changes in continent level had great effects on the restructuring of landscape and biota in this supercontinent (Montañez et al. 2013). In Brazil, the LPIA is recorded by deposits in the Paraná Basin, especially close to Itu city, São Paulo State. These deposits are known as “Itu rhythmites” or “Itu varvite” (Leonardos, 1938; Santos et al., 1996; Rocha-Campos, 2002). They are classified as a varve-type deposit. This rhythmite originates by the alternation of two sedimentation varve-types: (i) in cold periods continental ice retains sand and gravel-size sediments while clay and silt-size sediments in suspension in the water are deposited on the bottom of the lake by decantating, and, (ii) in warm periods, ice is melting and the sediments that were retained during the winter are now deposited. Classically, the Itu rhythmites are thought to have been deposited in a pro-glacial lake along the front of a glacier during the Late Paleozoic. The rhythmites are composed of alternating plan-parallel strata: light-colored, sandstone/siltstone layers (deposited during the warm periods by the action of dense turbidity currents) and dark colored, siltstone/mudstone layers (deposited during the cold periods, when the lake was frozen). The strata may have centimeter to millimeter scale, forming lithological couplets that were deposited annually, according to previous studies (Rocha-Campos, 2002; Franco et al. 2011).
At the top of the stratigraphic succession of the Itu rhythmites, various crinkled sedimentary surfaces were found. These structures concur with invertebrate ichnofossils, i.e. Cruziana d'Orbigny, 1842 and Diplichnites Dawson, 1873 (Fernandes & Carvalho, 2005, Lima et al., 2015). Whereas biomineralization processes in fresh sediments are well known (e.g. Lowenstam, 1981; Skinner, 2005; Konhauser & Riding; 2012), the detection of biominerals in the fossil record still remains a challenge. Distinguishing biominerals from inorganic mineral species or from diagenetic products is difficult. Although there are several studies about the deposition of the Itu rhythmites, there is still controversy regarding the origin, the paleoenvironment, and the periodicity recorded in the vertical extension of the outcrop.
In clastic aquatic deposits, MISS are common. These are sedimentary structures caused by benthic microorganisms interacting with physical sediment dynamics (Noffke et al, 1996; Gerdes et al, 2000, Noffke et al. 2001; Noffke 2010). These structures are very characteristic and cannot be mimicked by abiotic structures, especially, because they include fossil mat fabrics. Here, we present MISS structures in the Itu rhythmites throughout multimethod
approaches, such as petrological, geochemical and magnetic analyses. In addition, we present an interpretation that ancient microorganisms colonized the bottom of the lake and significantly influenced the paleoecology of bottom dwellers, implying in a modified depositional model of the Itu rhythmites.
Study area and geological setting
The Paraná Basin is an intracratonic basin that encompasses approximately 1.7x106 km2 of central-eastern South America. Six super-sequences are recognized in this basin: the Rio Ivaí (Ordovician/Silurian), the Paraná (Devonian), the Gondwana I (Carboniferous/Permian), the Gondwana II (Triassic), the Gondwana III (Jurassic/Cretaceous), and the Bauru (Cretaceous). The Gondwana I Super-sequence represents a transgressive-regressive cycle related to variations in sea level during the basin´s evolution (Milani et al., 2007). In this unit, the Tubarão and Passa Dois Supergroups occur. The Itu rhythmites belong to the upper part of the Tubarão Supergroup, specifically to the sub-unit called Itararé Group (Late Carboniferous to Early Permian). The Itu rhythmites are located in the central-eastern region of São Paulo State, approximately 90 km from the São Paulo city (Fig. 1).
Following palynological analyses, the Itu rhythmite was deposited during the late Pennsylvanian (Kasimovian/Gzhelian) age, within the Crucisaccites monoletus Interval Zone (Souza et al., 2010). According to Caetano-Chang & Ferreira (2006), the light layers have grain sizes of silt to sand, and are composed mainly of quartz grains cemented by calcite and silica, with rare feldspar and mica. The authors also described plan-parallel laminations, bioturbations and dropstones. Dark laminae are interpreted as shales (Santos et al. 1996; Caetano-Chang & Ferreira, 2006).
The Itararé Group records the Late Paleozoic Ice Age of the Gondwana Supercontinent (Santos et al., 1996). Its marine and continental sediments were deposited under glacial and periglacial climate conditions (Souza et al. 2010). Glacially controlled deposition in the Paraná Basin started in the Bashkirian (Early Pennsylvanian, Carboniferous), when ice moved into the basin, advancing up to 200 km. During the deposition of Itararé Group, the paleoenvironment consisted of a proglacial lake influenced by the ice streams of the Windhoek Ice Sheet - WIS (Santos et al., 1996). The last deposits of Itararé Group, as well as
the Guatá Group (overlying stratigraphic unit), record, however, a change of the glacial period (LPIA) to the warm period of Cisuralian (Vesely & Assine, 2006).
Figure 1. A) Paraná Basin (red) in the Gondwana supercontinent in the Lower Permian-Upper Carboniferous, ~360-270 Ma (modified from Veevers, 2004); B) Paraná Basin with the location of the Itu quarry, showing the Itu rhythmites, red star (modified from Milani, 1998); C) stratigraphic column of the Carboniferous-Permian interval, with the location of the Itu quarry (Mafra Formation, Campo Mourão Formation, Itararé Group (modified from Ianuzzi, 2010).
Material and methods
Samples
The collection of samples was conducted close to the “Parque do Varvito” (Varvito Park), in the Ituana quarry (23°16'6.20" S and 47°19'15.19" W), next to the urban area of Itu city (Fig. 1). This quarry presents around of 10 meters of exposure of rhythmites; the samples were collected along a perpendicular line crossing the upper 3 meters of the outcrop.
We used 32 samples in this study. Two representative samples were chosen for the compositional and magnetic analyzes (Table 1). All samples are deposited in the Laboratory of Paleoecology and Paleoichnology (LPP) of the Federal University of São Carlos (Brazil).
Table 1. Samples collected from the Ituana quarry, analysis and techniques applied to each of them.
sample (total= 32) macroscopic analysis thin section SEM/EDS TEM EPMA µ-XRF magnetic analysis Raman spectroscpy XRD LPP-0042 to 0072) x LPP-0041 x x x x x x LPP-0040 x x x x x x x LPP-0041 (extract) x LPP-0040 (extract) x x
Thin section analysis
Thin sections of 30µm were prepared (without a glass coverslip to allow compositional analyses). The thin sections were analyzed at the Laboratório de Paleohidrogeologia of State University of Campinas (UNICAMP), with a Carl Zeiss petrographic microscope Scope A1 ZEISS). The images were recorded with a ZEISS AxioCam camera and processed with ZEISS AxioVision® 4.8.2.0. (2006) software.
Material extraction
The sample preparation for scanning electron microscopy (SEM), transmission electron microscopy (TEM) and the rock magnetic analysis was performed at the Brazilian Synchrotron Light Laboratory (LNLS), using the LQI (Chemistry Laboratory) infrastructure. The process started with the milling of dark laminae, following by dissolution with ethanoic acid and sodium acetate. The ferromagnetic material was precipitated and extracted with a neodymium magnet. The extracted material was dried and weighed (protocol by Strehlau et al., 2014).
Electron Microprobe Analysis (EPMA), Scanning Electron Microscopy and Energy Dispersive Spectroscopy (SEM/EDS)
Compositional analysis, elemental maps and images were acquired with the SEM-FEG ENV (FEI Quanta) equipment at the Brazilian Nanotechnology National Laboratory (LNNano). The images were obtained using the Everhart Thornley SED, in secondary electron detection mode, with energy of 15 to 20 kV. The maps were performed using an Oxford Instruments EDS detector, operating the AZtec software. Additional images, quantitative data and cathodoluminescent (CL) images were collected using the JEOL 8530F electron probe at the Carnegie Institution for Science (Washington, DC). The probe was operated at 15 kV and 20 nA. Elemental maps were obtained using both WDS detectors and the Thermo Scientific Noran System 7 EDS system (NSS). Hyperspectral CL images were collected simultaneously using the Ocean Optics xCLent CL system. Individual quantitative analyses were also performed using the NSS system. Samples were coated in iridium to facilitate mapping and analysis of carbon.
µ-X-Ray fluorescence (µ-XRF)
µ-XRF analysis was performed using the beamline at the LNLS (Perez et al. 1999). The samples were cut in 4x2 cm size blocks and posteriorly polished. The beamline was used in micro-beam mode with the KB focusing system in order to reach a beam size of 12 x 25 mm diameter, at room temperature. The excitation was made in white-beam mode (3 – 14 keV). The data was treated and the elemental maps were obtained using the PyMca 4.6.0 software (developed by European Synchrotron Radiation Facility - ESRF).
Transmission Electron Microscopy (TEM)
The images were obtained using the JEOL JEM 2100 LaB6 TEM equipment at the Brazilian Nanotechnology National Laboratory (LNNano). The extracted magnetic material was allocated in a copper grid. The analyses were performed with the acceleration voltage of 200 kV and the resolution of 0.25 nm point-to-point.
Raman Spectroscopy
Raman imaging was performed with a WITec -Scanning Near-Field Optical Microscope that has been customized to incorporate confocal Raman spectroscopy imaging. The excitation source was a frequency-doubled solid-state YAG laser (532nm) operating between 0.01-5 mW output power. Optical microscopy images were captured by a camera system attached to the microscope. Objective lenses that were used included a 20x long working distance (LWD) for large area scans and a 100x oil immersion lens for small area scans, with a 25 m optical fiber acting as the confocal pinhole. Spectra were collected on a Peltier-cooled Marconi 40-11 CCD chip, after passing through a f/4 300mm focal length imaging spectrometer using a 600 lines/mm grating. The instrument produces Raman images by accumulating a Raman spectrum at each image pixel as the sample stage translates. Raman peaks of interest were then chosen, and their occurrence throughout the image computed by using a Gaussian fit to the data. WITec Project Plus software was used to map peaks of interest across the sample and compute peak intensity maps.
X-ray diffraction
X-ray diffraction analysis was performed using the XRD2 beamline at the LNLS. The data were obtained withexcitation energy of 7 keV, with beam size in order of ~0.5 mm x 1.5 mm (V x H). The energy was chosen to be bellow Fe K-edge, and avoiding the spurious signal from the Fe X-ray fluorescence. The grazing incidence (GID) geometry is proposed in order to limit the depth of penetration into the sample, to isolate the signal from superficial laminae. The Linear Mythen (Detrics) detector, 1 k, was utilized to collect the diffraction data. The samples were cut into small blocks 1 to 2 cm2 in size and positioned on the goniometer without further manipulation. The analyses were performed in both the dark laminae and light layers.
Rock magnetism
Rock magnetism measurements were performed at the Laboratório de Paleomagnetismo of University o São Paulo (USPMag) and University of Brasilia, Brazil. Acquisition of isothermal remanent magnetization curves (IRM), hysteresis loops, first order reversal curves (FORC) and low-temperature magnetization measurements were performed to determine the magnetic properties of the studied bulk sample and a sample from extracted magnetic material. Hysteresis loops, IRM acquisition curves and FORC diagrams were determined at room temperature. Measurements were performed with applying fields of up to 1 T using a Princeton Measurements Corporation Micromag vibrating sample magnetometer (VSM). Saturation magnetization (Ms), saturation remanent magnetization (Mrs), coercivity (Bc), and coercivity of remanence (Bcr) are all determined from hysteresis and backfield measurements. The ratios of Mrs/Ms and Bcr/Bc reflect relative trends in grain-size distribution (e.g., Day et al., 1977; Dunlop, 2002a; 2002b). However, these standard hysteresis parameters provide only a measure of the bulk magnetic properties and therefore are not suitable for discriminating the different magnetic components contributing to the magnetization in samples with mixed or more complex magnetic assemblage. Given the complex magnetic mineralogy in our samples, we used FORC diagrams to identify and discriminate the different magnetic mineral grains (Roberts et al., 2014). FORC measurements were performed at room temperature after 586 reversal curves with an averaging time of 200 ms. FORC diagrams were calculated using the FORCinel software package (Harrison and Feinberg, 2008) using a smoothing factor of 15 for all samples. In addition, low-temperature magnetization measurements were carried out using a Magnetic Property Measurement System XL (Quantum Design) at the University of Brasilia. These measurements were performed in order to identify the transition phases between different magnetic fractions, in particular the Verwey (~120 K) and the Morin (~260 K) transitions for stoichiometric magnetite and hematite, respectively. Measurements of Room Temperature Saturation of IRM (RTSIRM) and Field-Cooling, Zero-Field Cooling (FC/ZFC) curves were carried out on representative sample. Samples were cooled to 10 K in both zero field and 5 T, respectively. At 10 K, a 5 T field was applied and was then switched off to import a RTSIRM and the MPMS was reset. FC/ZFC curves were measured during warming in zero field in scan mode at 5 K/min.
Results and interpretations
Field data
The quarry of Itu rhythmites presents alternating dark and light layers forming couplets. These couplets can reach an average thickness of 45 cm at the bottom of the quarry; they thin upward to only 1 to 2 mm at the top. The light layers are in average 50 to 80% thicker than the dark ones, whereas the thickness of the dark laminae remains constant. Only towards the top of the succession do the light and dark laminae have the same thicknesses.
At the top of the quarry, low profile ripple marks, as well as wrinkle structures and sinoidal cracks occur on bedding surfaces (Fig. 2). In vertical section, dark laminae become visible. The ripple marks show crest-to-crest distances of 1.5 cm, but have only a height of up to 2 mm; the wrinkle structures cover 9 to 108 cm2 areas and show mm-scale, irregular crinkles of no preferred orientation. The cracks have 15 to 20 mm lengths and are weathered to a depth of < 1 to 2 mm. Ichnofossils are associated, commonly preserved as positive epirelief. The tracks range from 3 to 10 mm, and have sometimes a slight sinuosity. They remain surface parallel. There are no podial imprints. Fig. 3 A shows a vertical section through a domal structure. We interpret the wrinkle structures, the low-profile ripple marks and sinoidal cracks as in situ preserved microbial mats forming MISS. The wrinkle structures were caused by the mechanical stress of invertebrate movement ‘bulldozing’ through the coherent microbial mat (Fig. 2 A and sketch in 2 B). The ancient microbial mat was thick enough to cover the proceeding ripple marks like a cloth, so the sediment surface became almost planar. Sinoidal cracks record the episodic desiccation of microbial mats. The domal structure in Fig. 3 A is interpreted as gas dome, once developed beneath a sediment-sealing microbial mat.
The ancient microbial mats developed on the bottom of the proglacial lake, and were episodically subaerially exposed along the shore lines. The substrate was ideal for the proliferation of foraging invertebrates.
Figure 2. MISS from the Itu rhythmites: A) wrinkle structure caused by the invertebrate movements disturbing the coherent microbial mat. Proceeding ripple marks were completely smoothened by the microbial mat. B) sketch for illustration. C and D) cracks caused by contraction and rupture of the microbial mats during episodic subaereal exposition. E) modern microbial mat from Lagoa Vermelha (Rio de Janeiro, Brazil), showing cracks comparable to the studied samples from Itu rhythmites.
Petrology
In vertical section through the MISS, the alternating dark and light layers reveal differences in grain sizes (light layers have grains of silt/sand size whereas dark laminae have grains of mud/silt size). The dark laminae include copious amounts of amorphous organic matter (kerogen) and iron oxide (almost 95%). Oriented quartz grains in the dark layers make approximately 5% of the matrix. The light layers consist of 92% of silt and 8% mud. Some laminations include silica cement. The main mineral in theses layers is quartz (approximately 90%), in addition to rare feldspar (4%). Also, pollen (1%) and iron oxide coating occur (up to 5%).
The vertical sections also reveal that the dark laminae form different MISS including gas domes, fenestrae fabrics, and sinoidal structures. The millimeter-scale cavities beneath the fossil gas domes are partially or completely filled by silica cement (Fig. 3A). Undulated laminations are present, mainly towards to the bedding surface, Fig. 3B. Fenestral fabrics, characterized by elongated fenestrae pores ranging from 20 µm to 50 µm occur in the light-colored layers (Fig. 3C). Gas domes and fenestral fabrics commonly do occur together in areas covered by microbial mats: gas domes are local small domes that rise on the
depositional surface due to increasing gas pressure beneath a sediment-sealing microbial mat. The gases are also responsible for the formation of the pores, which are aligned parallel to the microbial mat layers. Some of the dark laminae form sinoidal structures of 2 to 5 mm heights (Fig. 3C). Sinoidal structures represent ripple marks that were overgrown by microbial mat and then buried. The high concentration of organic matter in the dark layers that build up the various MISS strongly supports the biological interpretation. In contrast, no MISS was detected in the white layers, which probably were formed without any microbial influence.
µ-X-Ray fluorescence
µ-XRF (X-ray Fluorescence) maps of elemental distribution were acquired from the surface-near areas of the samples. The maps showed that iron (Fe) is concentrated in the MISS-forming dark layers, whereas silicon (Si) and other elements that can be attributed to siliciclastic material are distributed in the light layers (Fig. 3D). Titanium (Ti) is distributed in the entire samples, but has a slightly higher concentration in the dark laminae (Fig. 3E). Calcium (Ca) is concentrated only in the light layers.
The elemental composition of the light layers is consistent with the clastic origin; whereas the high concentration of Fe in the dark laminae supports a biogenic origin of Fe deposition (see also later).
Figure 3. A) thin section (sample LPP-0040) showing the dark laminae (DL) and light layers (LL) and the gas domes (GD) filled by silica cement between the dark laminae; B) thin section (sample LPP-0041) showing the undulating laminations in the top-most layers (towards to the top bed surface) and the intercalation between the dark laminae (DL) and light layers (LL); C) thin section (sample LPP-0040) showing the difference in the grain size in the dark laminae (DL) and the light layers (LL), quartz grains as the major component in the LL and organic matter (om) in the DL, laminoid-fenestral fabrics (ff) and sinoidal structures (yellow arrow) in the DL; D) µ-XRF image with elemental map showing the distribution of Fe in the dark laminae and the Ca and Si in the light layers; E) µ-XRF elemental map with the Ti distributed mainly in the dark laminae, and the Ca in the light layers.
SEM/EDS and TEM analysis
In SEM/EDS analyses, phyllosilicates (including Si, Al and K) were detected in both the light layers and in the dark laminae. In the dark laminae, the quartz and feldspar grains show a smooth surface, which we interpret as fossil biofilm (Fig. 4A). Magnetic material extracted from the putative biofilm was analyzed by EDS and showed enhanced concentration of Fe (67 to 80%) and oxygen (O) (23 to 26%). Minor percentages of C, Al, Si and Ti occur (Fig. 4B). In energy dispersive X-ray spectroscopy (EDS), C and O were the main constituents of these putative biofilms (Fig. 4C). Transmission electron microscopy (TEM) images of the putative biofilm suggests elongated hexaoctahedral magnetite nanoparticles from 200 to 250 nm of length (Fig. 4D); the extracted material suggests spheroidal nanoparticles of iron minerals (most likely magnetite, according magnetic analyses) with approximately 30 nm diameter, some of them with octahedral habit, occur as well (Fig. 4E).
We interpret the data obtained from the dark laminae as indicative for fossil biofilms. The biofilm fossils and the iron composition of the magnetic material extracted from these biofilms supports a bio-precipitation of the iron minerals. The phenomenological TEM textures suggest a participation of magnetotactic bacteria (MTB) in the iron deposition.
Figure 4. A) SEM/EDS analysis in the surface of dark layer showing grains possibly covered by fossil biofilm including EPS; B) SEM/EDS analysis in the extracted magnetic material and elemental map showing that the main composition is Fe and O, with some minor Cr, Ti and C; C) grains covered by a smooth texture with a composition compatible to that of modern EPS; D) putative elongated magnetite crystal related to magnetosome from magnetotactic bacteria; E) hexoctahedral nanocrystals of magnetite.
Raman spectroscopy
Raman spectroscopy (Fig. 5) revealed spectral peaks corresponding to anatase (TiO2) shifts at 143, 399, 513 and 639 cm-1. Also, quartz (201, 263 and 467 cm-1) and feldspar (sanidine, at 154, 284, 476 and 512 cm-1) were detected. The Raman map of a selected area in the sample showed that the quartz and feldspar are distributed in both dark and light layers. Although the anatase is distributed in both dark and light layers, it occurs in a higher concentration in the dark ones. Anatase distribution here is in alignment with the biofilm detected in these laminae, as well the iron detected in the µ-XRF and SEM/EDS analysis. Carbon shows the same distribution pattern as the anatase (peaks at 1351 and 1589 cm-1). This distribution pattern suggests a bio-precipitation of the anatase.
Figure 5. Raman spectroscopy analysis in an area of 1 x 2,5 mm of the sample (LPP-0041). Elemental map shows the distribution of quartz (yellow), anatase (blue), carbon (red) and feldspar (green). In the light layers quartz grains predominate, while in the dark layers anatase and carbon predominate. Raman spectra with signature peak shifts for anatase at 143, 399, 513 and 639 cm-1; for kerogen at 1351 and 1589 cm-1; for feldspar (sanidine) at 154, 284, 476 and 512 cm-1 and for quartz at 201, 263 and 464 cm-1.
X-Ray diffraction
The X-ray diffraction analyses were performed on the polished surface of a small block of sample (IcI-009), in both the dark laminae and light layers (Fig. 6A). In the dark laminae, the following minerals were detected: muscovite, calcium phosphate, rutile and anatase (Fig. 6B). The most intense anatase Bragg reflection (2 at 29.26º) appears convoluted with the most intense peak of calcium phosphate (Ca(PO3)2) (~28.99º), what hinders the definitive identification. However, the second most intense peak of anatase phase, which appears around 55.96º, is one more evidence that this phase of this mineral is present is the material. Then, this set of evidences plus the Raman data make a strong indicative of the presence of anatase in the dark laminae. In the light layers it was possible to acquire the diffractogram of SiO, in the form of alpha-quartz, as expected (Fig. 6C). The reference code and other standards specifications are shown in Table 2.
Figure 6. A) Analyzed sample (LPP-0040) showing the dark laminae and light layer; B) diffractogram from the dark laminae, showing the mineral phases muscovite, calcium phosphate, rutile and anatase; C) diffractogram from the light layer, showing the alpha-quartz mineral phase.
Table 2. Reference diffraction pattern of crystalline inorganic phases identified in the dark and light layers, through the application of X-ray diffraction, excited with 7 keV.
Mineral Reference code (ICDD*) Mineral name PDF index name/ ICDS name
Empirical formula Chemical formula
Muscuvite 00-007-0042 Muscovite-3T Potassium Aluminum Silicate Hydroxide Al2.90H2KO12Si3.10 (K, Na)(Al, Mg, Fe)2 (Si3.1Al0.9) O10 (OH)2 Calcium phosphate 00-009-0363 Calcium Phosphate Calcium
Phosphate CaO6P2 Ca(PO3)2
Rutile 01-088-1174 Rutile - synthetic Titanium Oxide O2Ti TiO2
Anatase 01-083-2243 Anatase, syn Titanium Oxide O2Ti TiO2
*ICDD – International Centre for Diffraction Data
Rock magnetic analysis
Rock magnetic analysis of the samples was performed mainly in the dark laminae (see Fig. 7). The IRM acquisition curve (Fig. 7A) and hysteresis loop of bulk samples (Fig. 7B) show two different magnetic phases, one with a low coercivity phase and the other with a high coercivity phase, probably magnetite and hematite respectively. In this case, the low coercivity phase presents the major contribution for the magnetization. The FORC diagram for the bulk sample (Fig. 7C) shows a slight central distribution. Low temperature data (FC/ZFC curves) shows that the Verwey transition (Tv) has two distinct peaks at 100 and 120 K (Fig. 7D).
The IRM acquisition curve (Fig. 7E) and hysteresis loop (Fig. 7F) for extracted magnetic material show higher values in comparison with the bulk sample. This indicates that extracted material is dominated by low coercivity minerals. The FORC diagrams (Fig. 7G) show a
distribution that is coherent with minute magnetite crystals, but we cannot observe the central ridge distribution typically observed for biogenically produced magnetite.
The low temperature magnetic results suggest that part of the magnetic mineralogy is composed of abiogenic magnetite (peak at 120 K) that can be interpreted as having a detrital origin. Nevertheless, the 100 K peak of Verwey transition indicates that some fraction of the abiogenic iron oxides was transformed into biogenic magnetite (Chang et al. 2016). Although there were episodes of anoxic conditions for the microorganisms after burial by sediments, there is no magnetic signal of sulfur minerals such as pyrite or greigite. The absence of these minerals may exclude the possibility of sulfate reduction activity (in the formation of magnetite), corroborating the magnetic analysis that also did not show any magnetic signature of iron sulfide.
Figure 7. Magnetic analysis in the bulk sample and in the extracted magnetic material. A) isothermal remnant magnetization curves (IRM) of the bulk sample; B) hysteresis loop of the bulk sample; C) first order reversal curves (FORC) diagram of the bulk sample, showing a slight central ridge distribution typical of biogenic magnetite; D) low-temperature magnetization measurements after zero-field cooled (ZFC) and field-cooled (FC) conditions (FC/ZFC curves), showing the Verwey transition in peaks at 100 and 120 K; E) isothermal remnant magnetization curves (IRM) of the extracted magnetic material; F) hysteresis loop of the extracted magnetic material; G) first order reversal curves (FORC) diagram of the extracted magnetic material without the central ridge distribution related to the biogenic magnetite, although it indicates a very small crystal size.
Discussion
Microbially induced sedimentary structures in the Itu Rhythmite
The dark laminae have been interpreted as mudstones (Rocha-Campos, 2002) or shale (Caetano-Chang & Ferreira 2006). However, the analyses presented here show a significant presence of organic matter in the dark laminae. Iron minerals and anatase in combination with fossil bacterial EPS are preserved.
Different macroscale MISS including wrinkle structures, levelled ripple marks, sinoidal structures, and gas domes point towards a dense colonization of the lake bottom by benthic microbiota. In analogous glacial deposits located in Mafra Formation (Southern Brazil), Netto et al. (2008) observed microbially-induced wrinkle structures that resemble strongly those presented here from the Itu rhythmites. Also observed was the common occurrence of these structures with ichnofossil tracemakers (D. gouldi), suggesting a narrow link between them. Netto et al. (2008) associated the specific deposition over the microbially-induced wrinkle structures with periodic drainage of some shallow lakes, which can create freshwater marshes that may have partially dried up in some periods (Netto et al. 2008).
As shown in the SEM/EDS analysis, the smooth biofilm surface that enveloped feldspar and quartz grains, plus the carbon detected in these envelopes, point to EPS fossilized in the dark layers. The EPS may be the major contributor to organic carbon in sediments (Decho, 2000). The presence of iron primarily in the dark MISS laminae is remarkable. This iron presented most likely a magnetic behavior compatible to biogenic magnetite, although there are two different magnetic phases in the dark layers: one with high coercivity (hematite) and other with low coercivity (attributed to magnetite). Results of low temperature measurements suggest that part of low coercivity phase, i.e. magnetite, is formed by biogenic magnetite.
Hypotheses for a microbially-induced Fe and Ti minerals precipitation
In µ-XRF maps, the iron is distributed plane-parallel in the dark laminae. Microscopically, the iron is generally found in alignment with biofilm, located in areas of organic matter in high concentration. In other areas outside the dark laminae, as in the light layers, there is little or no iron. This suggests a relation between the biofilms and iron precipitation. Many microorganisms reduce or oxidize iron in their metabolism (Konhauser & Riding, 2012). Magnetotactic bacteria (MTB) can precipitate iron in a biologically controlled way, forming magnetosomes, which allow the bacterial cells to align themselves along the magnetic field and to reach the optimal position in the water column with respect to chemical gradients (Blakemore, 1975). The processes of iron biomineralization can influence the crystallographic and magnetic properties of these minerals (e.g. Vali & Kirschvink, 1991, Thomas-Keprta et al., 2000; Abrajevitch et al., 2016), allowing us to differentiate between these minerals according to their origin (biogenic or inorganic precipitation), using techniques such as X-Ray diffraction (Thomas-Keprta et al. 2000) or rock magnetic techniques (Egli, 2004). Here, we applied the magnetic techniques to detect the biogenicity of iron minerals in the dark laminae. In the modern glacial lakes of Clearwater, Silver and others in the Canadian province of Ontario (Fortin et al., 1993), microbial mats are produced by iron bacteria. Ferrous ions in the surrounding water may foster the development of iron-metabolizing microbes (Fortin et al., 1993; Schieber and Glamoclija, 2007). Iron oxidizing bacteria, such as Gallionella and
Leptothrix, can oxidize Fe(II) according the reaction Fe2+ + 0.25O2(aq) + 2.5H2O → Fe(OH)3(s)
+ 2H+ (Schieber and Glamoclija, 2007). Iron-reducing bacteria can use the Fe ions from ferrihydrite (Fe2O3.0,5H2O), the most common bio-available form of Fe(III) oxide in nature. The magnetite particles may be a result of the reaction during the dissimilatory iron reduction through a coupled biotic-abiotic process (Hansel et al. 2003). One of these reactions is the reduction of iron oxide to nanocrystals of ferromagnetic magnetite by the action of hyperthermophilic iron reducing bacteria (Lovley et al., 1987).
Another hypothesis for the origin of magnetite in the dark layers is related to MTB. These microorganisms preferentially live under specific redox conditions in the oxic-anoxic transition zone (Bazylinski and Moskowitz, 1997) and present octahedral crystals of magnetite arranged in chains inside their cells, the magnetosomes. These crystals range from 19 to 136 nm in length and 14 to 112 nm width (Lins & Farina, 1998). When the bacteria decease, their magnetosomes can be preserved in the rock record, but diagenetic alteration and degradation of organic matter can disarrange the chain of the magnetosomes (Kopp &
Kirschvink, 2008, Amor et al. 2014). In our study, TEM analyses showed isolated octahedral crystals. They are not arranged in chains. However this disarray may be an artefact of the destructive ferromagnetic mineral extraction process which includes mechanical crushing and extraction with acid. Elongated magnetite crystals may be related to specific magnetofossils, such as those originated from MTB found in a borehole in Quaternary sediments from the Atlantic Coastal Plain of New Jersey (Schumman et al. 2008). The crystals found in dark layers are approximately 200 to 250 nm in length. Schumman et al. (2008) describe their longest magnetofossil as 580 nm. According to the same authors, other modern examples derive from the Bahamas are up to 170 nm in length. According to the authors, such magnetosomes occur as isolated features or as aggregates of originally isolated crystals; this is in contrast with most magnetosomes of MTB, which are arranged in chains. Although the possible existence of magnetofossils should not be excluded, the evidence for this occurrence in TEM images is not sufficient to affirm that there was an expressive participation of MTB in the iron deposits of the dark laminae.
The anatase (TiO2) distribution concurs with the biofilm texture, a fact that suggests the possibility of this mineral resulting from bio-precipitation. Bower et al. (2015), in incubation experiments of cyanobacteria in sandy environments, obtained anatase bioprecipitation under simulated conditions similar to early diagenesis.
Ichnofossils
The invertebrate ichnofossils found in the surface of the dark laminae are very well preserved, which allows us to observe important details for identification. The good preservation of the ichnofossils is in part due to the fine-grained nature of the dark sediment, but also because of the EPS of the biofilms. According to Fernandes et al. (2005), two ichnogenus are abundant in the rocks of the Itararé Group: Isopodichnus Bornemann (1889) and Diplichnites Dawson (1873) possibly attributed to arthropods myriapods (Draganits et al., 2001). The fossil traces preserved in positive epirelief indicate the presence of these myriapods.
The wrinkle structures occur where apparently some arthropod has moved under the microbial mat, causing dragging and distortion of the mat without breaking. This lead to the preservation of trace fossils in high relief forms.
The depth of the lake was variable during the final time of deposition. Cracks in the dark laminae are evidences of subaerial exposure of the lake bottom and indicates that there may have been periods of total water shortage in the system. The shallow water shortly before and
