UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE PESQUISA EM BIOENERGIA unesp PROGRAMA INTEGRADO (UNESP, USP E UNICAMP) DE PÓS-GRADUAÇÃO EM BIOENERGIA Green stabilization of nanoscale zero valent iron (nZVI) with rhamnolipids produced by agro-industrial waste: application on nitrate reduction Cinthia Cristine de Moura Tese apresentada ao Instituto de Pesquisa em Bioenergia de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências. Orientador(a): Jonas Contiero Setembro - 2019 Green stabilization of nanoscale zero-valent iron (nZVI) with rhamnolipids produced by agro-industrial waste: application on nitrate reduction CINTHIA CRISTINE DE MOURA Rio Claro 2019 Tese apresentada ao Instituto de Pesquisa em Bioenergia de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências. Orientador(a): Jonas Contiero M929g Moura, Cinthia Cristine de Green stabilization of nanoscale zero valent iron (nZVI) with rhamnolipids produced by agro-industrial waste : application on nitrate reduction / Cinthia Cristine de Moura. -- Rio Claro, 2019 137 f. : il., tabs. Tese (doutorado) - Universidade Estadual Paulista (Unesp), Instituto de Pesquisa em Bioenergia - IPBEN, Rio Claro Orientador: Jonas Contiero 1. Biossurfactantes. 2. Nanopartículas de ferro zero valente. 3. Resíduos agrícolas. 4. Remediação Ambiental. 5. Design experimental. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca do Instituto de Biociências, Rio Claro. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. User Máquina de escrever Título alterado para "Green stabilization of nanoscale zero-valent iron (nZVI) with rhamnolipids produced by agro-industrial waste: application on nitrate reduction" “A lei da mente é implacável. O que você pensa, você cria; O que você sente, você atrai; O que você acredita Torna-se realidade”. Buda AGRADECIMENTOS Primeiramente gostaria de agradecer a Universidade Estadual Paulista “Júlio de Mesquita Filho” e ao Instituto de Pesquisa em Bioenergia, bem como aos coordenadores e professores. À Capes pela bolsa de auxílio financeiro. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Código de Financiamento 001. Agradeço ao Prof. Dr. Jonas Contiero pela sua sabedoria e paciência, que me ensinaram muito. Ao Prof. Dr. Miguel Jafelicci e ao Prof. Dr. Rodrigo Marques por gentilmente cederem o laboratório no Instituto de Química da Unesp de Araraquara para realização dos testes com as nanopartículas. Aos meus pais Júlio e Sandra por sempre incentivarem a continuidade deste trabalho, por me ouvirem e por sempre estarem ao meu lado nos momentos difíceis. Ao meu irmão Douglas, a minha cunhada Mariana e meu sobrinho Diogo, por trazerem alegria nos poucos momentos que estávamos juntos. Aos amigos do LMI, pelo convívio diário, pelos papos na hora do café, por me ensinarem, por me ouvirem, aconselharam, e por terem sido minha família nesses 4 anos. Ao Rodolfo e Caio que me ensinaram e ajudaram na síntese das nanopartículas e nas discussões dos resultados. A Ana Maria, que foi minha chefe, minha estagiária, minha mãe, minha irmã, minha filha e em todas as situações: minha amiga. Nada disso teria sido possível sem você ao meu lado. A Thalita Moura e Yaliana Tafurt, mesmo longe nunca deixaram de me apoiar. A Cristhian Lao, que me apoiou, ouviu e ajudou durante todo o doutorado. Obrigada por sempre estar ao meu lado, por não desistir de mim e nem me deixar desistir. As minhas primas, que considero como irmãs: Flávia Nunes e Nathália Giovanni. Ao meu tio Fernando que me deu várias caronas, os longos papos no transito faziam as viagens mais divertidas. Aos meu pais, tia Elaine, tia Rosana, tio Fernando, primas Rafaela e Nathália, obrigada por terem vindo na defesa, obrigada por encararem as longas horas de viagem e por fazerem parte desse momento tão especial! E agradeço a todos aqueles que de alguma forma contribuíram para o andamento do trabalho realizado durante esta etapa da minha formação profissional. Resumo A contaminação ambiental causada por compostos orgânicos é um importante problema que afeta solos e água superficiais. Para reduzir ou remover esses poluentes, os locais contaminados são geralmente tratados com métodos físicos e químicos. No entanto, a maioria dessas técnicas de remediação é custosa e geralmente leva à remoção incompleta e à produção de resíduos secundários. A nanotecnologia consiste na produção e aplicação de estruturas extremamente pequenas, cujas dimensões estão na faixa de 1 a 100 nm, neste cenário a nanopartícula de ferro zero valente representa uma nova geração de tecnologias de remediação ambiental. É não tóxica, abundante, barata, fácil de produzir, e seu processo de produção é simples. No entanto, a fim de diminuir a tendência de agregação, a nanopartícula de ferro zero é frequentemente revestida com surfactantes. A maioria dos surfactantes é quimicamente sintetizado a partir de fontes petroquímicas, eles são persistentes ou parcialmente biodegradáveis, enquanto oferecem baixos riscos à saúde humana, esses compostos podem prejudicar plantas e animais. Para diminuir o uso de métodos químicos, a síntese e estabilização verde de nanomateriais metálicos apresentam-se como uma opção menos perigosa ao meio ambiente. Os biossurfactantes podem potencialmente substituir qualquer surfactante sintético, eles são compostos extracelulares produzidos por microrganismos, como bactérias, e cultivados em diferentes fontes de carbono, podendo ser substratoshidrofílicos. Os biossurfactantes possuem uma grande variedade de estruturas químicas e propriedadesde superfície e entre eles estão os ramnolipídios que já foram intensamente investigados e estudados. Os ramnolipídios podem ser produzidos pela Pseudomonas aeruginosa em diferentes substratos, incluindo o glicerol. Uma produção bem-sucedida de biossurfactantes depende do uso de materiais renováveis e de baixo custo. O glicerol bruto, principal subproduto do processo de transesterificação em uma usina de biodiesel, é um substrato amplamente utilizado para a produção de ramnolipídios. A fim de obter a melhor temperatura e concentração inicial de glicerol, o design rotacional composto central e o método de superfície de resposta foram empregados para delimitar as melhores condições para aumentar a produção de ramnolipídeos e diminuir o glicerol remanescente no meio. Com o auxílio do Método de Superfície e Resposta foi possível verificar a viabilidade do uso do glicerol bruto livre de sal, atingindo uma produção de 2,63 g/L de ramnolipidios e depleção total da fonte de carbono, no meio otimizado contendo 25 g/L de fonte inicial de carbono a 32 ° C. Em seguida, as nanopartículas de ferro zero foram sintetizadas utilizando a redução química com borohidreto de sódio. Foram testadas duas metodologias: (i) adição de ramnolipídios durante a síntese química e (ii) adição após a síntese. As nanopartículas foram subsequentemente testadas quanto à eficiência na redução de nitrato em água subterrânea simulada sob condições anaeróbias em pH 4. A nanopartícula de ferro zero sintetizada adicionando ramnolipídios após a síntese, mostrou a melhor eficiência com uma taxa de remoção de cerca de 78% de remoção de nitrato e concentração inicial de nitrato de 25. mg/L. O método para preparar nanopartícula de ferro zero, usando ramnolipídios como agente estabilizador, mostrou-se uma alternativa promissora para a funcionalização de superfície da nanopartícula, em substituição a surfactantes sintéticos e tóxicos Palavras chave: Biossurfactantes. Química verde. Design experimental. Glicerol. Nanopartícula de ferro zero valente. Águas subterrâneas. Abstract Environmental contamination caused by organic compounds is the most important challenge that affects a huge number of soils and water surfaces. To reduce or remove these pollutants, contaminated sites are usually treated using physical and chemical methods. However, most of these remediation techniques are expensive and commonly lead to incomplete removal and to the production of secondary wastes. Nanotechnology is the production and application of extremely small structures, whose dimensions are in the range of 1 to 100 nm and Nanoscale zero-valent iron represents a new generation of environmental remediation technologies, is non-toxic, abundant, cheap, easy to produce, and its reduction process requires little maintenance. Nonetheless, in order to diminish the tendency of aggregation, nanoscale zero-valent iron is often coated with surfactants. Most surfactants are chemically synthesized from petrochemical sources, they are slowly or partially biodegradable, while offer low harm to humans, such compounds can influence plants and animals. To decrease the use of chemical methods green synthesis and stabilization of metallic nanomaterials viable option. Biosurfactants can potentially replace virtually any synthetic they are extracellular compounds produced by microbes such as by bacteria and grown on different carbon sources containing hydrophobic/hydrophilic substrates. The biosurfactants have a wide variety of chemical structures and surface properties and among them is the rhamnolipids which have been intensively investigated and extensively reviewed, they can be produced by Pseudomonas aeruginosa from different substrates, including glycerol. Successful production of biosurfactants depends on the use of renewable materials and low cost. Crude glycerol is the primary byproduct of the transesterification process in a biodiesel plant and it is a widely used substrate for rhamnolipid production. To provide the best temperature and initial concentration of glycerol the central composite rotational design and response surface method were employed to increase rhamnolipids yield and lower the glycerol remaining in the medium. The response surface method methodology indicated the viability of the use of crude glycerol, reaching a production of 2.63 g/L of biosurfactant and total depletion of carbon source, at the optimized medium containing 25 g/L of initial carbon source at 32 °C. Then, the iron nanoparticles were synthesized using the chemical reduction of ferric ions with sodium borohydride. Were tested two methodologies: (i) adding rhamnolipids during the chemical synthesis and (ii) adding after the synthesis. The nanoparticles were subsequently tested for their efficiency in nitrate reduction in simulated groundwater under anaerobic conditions at pH 4. The nanoscale zero-valent iron synthetized adding rhamnolipids after the synthesis showed the best efficiency with a removal rate about 78% and initial nitrate concentration of 25 mg/L. The method for preparing nanoscale zero-valent iron using rhamnolipids biosurfactants as stabilizer was found as a promising alternative for the synthesis and surface functionalization of iron nanoparticles, in replacement to toxic synthetic surfactants Key words: Biosurfactants. Green chemistry. Experimental design. Glycerol. Nanoscale zero-valent iron. Groundwater. Lista de Figuras Figure 1.1 Illustration of the top-down and bottom-up approaches _______________ 19 Figure 1.2 Typical production of biosurfactants during growth __________________ 23 Figure 1.3 The relationship between biosurfactant concentration, surface tension, and formation of micelles. __________________________________________________ 24 Figure 1.4 Structure of the four main counterparts of rhamnolipids ______________ 28 Figure 1.5 Transesterification reaction of a triacylglycerol with an alcohol. ________ 33 Figure 2. 1 Schematic illustration of (a) surface modification and (b) network stabilization 52 Figure 2. 2 Molecular structure of the surfactants Sodium Dodecyl Sulfate (SDS), Alkylphenol polyethoxylate (Triton X-100), Polyvinylpyrrolidone (PVP), Polyethylene glycol (PEG) 54 Figure 2. 3 Molecular structure of the main biosurfactants (a) Sophorolipids, (b) Mannosylerythritol Lipids (MEL) (c) Emulsan (d) Surfactin (e) Rhamnolipids 63 Figure 2. 4 Application of nZVI for in situ remediation. 68 Figure 2. 5 The core consists of principally zero-valent iron and supplies force for the reducing reactions with pollutants. The shell is mostly iron oxides/hydroxides created by the oxidation of zero-valent iron and it supplies sites for chemical reduction reaction.⠀ 70 Figure 3. 1 Experimental rhamnolipids concentration plotted against rhamnolipids concentration predicted by the fitted model _________________________________ 98 Figure 3. 2 Experimental glycerol remaining concentration plotted against glycerol remaining concentration predicted by the fitted ______________________________ 99 Figure 3. 3 Response surface curve of interaction of initial glycerol and temperature (a) rhamnolipids; (b) glycerol remaining _____________________________________ 100 Figure 4. 1 FTIR spectra of rhamnolipids _________________________________ 112 Figure 4. 2X-ray diffraction peaks associated with nZVI particles after the synthesis (e) bare nZVI, (c) nZVI-A (a) nZVI-S. and associated with nZVI particles 1 month after the synthesis (f) bare-nZVI(d) nZVI-A and (b) nZVI-S _________________________ 113 Figure 4. 3 Zeta potential and pzc of nZVI ________________________________ 114 Figure 4. 4 Average particle diameter size distribution of nZVI particles. ________ 115 Figure 4. 5 FEGSEM images (magnitude 30.0 k) of (a) bare-nZVI (b) nZVI-A (c) nZVI- S _________________________________________________________________ 116 Figure 4. 6 TGA and DTA curves for (a) bare-nZVI, (b) nZVI-A and (c) nZVI-S. _ 117 Figure 4. 7 Effect of time and initial nitrates concentration on nitrates reduction using nZVI at pH 4 (a) 25 mg/L NO3-N (b) 50 mg/L NO3-N (c) 100 mg/L NO3-N and effect of time and initial nitrates concentration on ammonia concentration using nZVI at pH 4 (d) 25 mg/L NO3-N (e) 50 mg/L NO3-N (f) 100 mg/L NO3-N ________________ 121 Figure A. 1 Results of tests using P. aeruginosa strains in different temperatures using 25 g/L of glycerin regarding to (a) rhamnolipids production by P. aeruginosa PAO1, (b) rhamnolipids production by P. aeruginosa LBI (c) rhamnolipids production by P. aeruginosa __________________________________________________________ 133 Lista de Tabelas Table 1.1 Type and microbial origin of biosurfactants ________________________ 25 Table 1.2 P. aeruginosa strain in various carbon source._______________________ 27 Table 1.3 Production of Rhamnolipids by Pseudomonas strains using Glycerol _____ 35 Table 2. 1 Production of biosurfactants using different strains and carbon sources ...... 59 Table 2. 2 Remediation applications for most common pollutants 69 Table 3. 1 Real and coded values of independents variables for the central composite rotational design. _____________________________________________________ 95 Table 3. 2 Planning matrix for central composite rotational design and experimental results ______________________________________________________________ 96 Table 3. 3 Analysis of variance for response surface quadratic model regarding rhamnolipids. ________________________________________________________ 97 Table 3. 4 Analysis of variance for response surface quadratic model regarding glycerol remaining ___________________________________________________________ 99 Table 3. 5 Planning matrix for validation of optmization design and experimental results __________________________________________________________________ 101 Table 4. 1 Stabilization methodology of nZVI ______________________________ 110 Table 4. 2 Regions of mass loss and gain (%) of nZVI-A and nZVI-S samples. ____ 118 Table 4. 3 Observed pseudo first-order rate coefficient of nitrate reduction with nZVI __________________________________________________________________ 120 Table A. 1 Matrix of pre-test analysis with P. aeruginosa strains in different conditions. __________________________________________________________________ 130 Table A. 2 Pre-tests results for different pH, temperaturature and initicial concentration of glycerol pro-analysis _______________________________________________ 132 Table A. 3 Yield coefficients regarding to biomass yield on substrat (YX/S), rhamnolipid yield on substrat (YP/S) and rhamnolipid yield on biomass (YP/X). 134 Sumário 1. INTRODUCTION 13 1.1. NANOTECHNOLOGY 15 1.1.1. NANOTECHNOLOGY APLICATIONS 15 1.1.2. TYPES AND APPLICATIONS OF NANOPARTICLES: 16 1.1.3. PHYSICOCHEMICAL OF NANOTECHNOLOGY APPLIED TO THE STABILIZATION PROCESS 17 1.1.3.1. Superparamagnectic Nanoparticles 17 1.2. NANOSCALE ZERO-VALENT IRON (NZVI) 18 1.3. SYNTHESIS OF NON-STABILIZED NZVI PARTICLES 18 1.3.1. TOP-DOWN APPROACHES 18 1.3.2. BOTTOM-UP APPROACHES 19 1.3.2.1. Chemical Reduction 19 1.3.2.3. Electrochemical method 20 1.3.2.4. Pulsed Plasma: 20 1.3.2.5. Sonochemical Method 21 1.3.2.6. Biosynthesis of nzvi 21 1.4. SURFACTANTS 22 1.5. BIOSURFACTANT 23 1.6. PRODUCTION OF RHAMNOLIPIDS 26 1.7. PROCESS OF BIOREMEDIATION 29 1.8. RHAMNOLIPIDS IN BIOREMEDIATION 30 1.9. CRUDE GLYCEROL 32 2. Background and project relevance 35 3. Objectives 36 GENERAL 36 References 36 2. BIOSURFACTANTS AS GREEN CHEMICAL STABILIZERS FOR NANOSCALE ZERO VALENT IRON 48 Abstract 48 1. Introduction 48 2. Methods 50 3. Nanoscale zero-valent iron 50 3.1. AGGLOMERATION STABILIZATION 50 3.1.1. PRE-AGGLOMERATION STABILIZATION 51 3.2. STABILIZATION OF NANOSCALE ZERO-VALENT IRON 51 3.2.1. NETWORK STABILIZATION (OR VISCOUS STABILIZATION) 52 3.2.2. SURFACE MODIFICATION 52 Electrostatic repulsion 52 Steric stabilization 52 Electrosteric stabilization 53 3.3. CHEMICAL STABILIZATION AND ITS DISADVANTAGES 53 3.4. GREEN STABILIZATION AND ITS ADVANTAGES 56 4. Green stabilization allies: biosurfactants 58 4.1. HIGH MOLECULAR WEIGHT POLYMERIC BIOSURFACTANTS 60 4.1.1. EMULSAN 60 4.1.2. LIPOSAN 61 4.1.3. ALASAN 61 4.1.4. MANNOPROTEIN 61 4.2. LOW MOLECULAR WEIGHT BIOSURFACTANTS 62 4.2.1. FATTY ACIDS AND PHOSPHOLIPIDS 62 4.2.2. LIPOPEPTIDES 62 4.2.3. GLYCOLIPIDS 62 Rhamnolipids 63 Sophorolipids 64 Trehalose lipids 64 Mannosylerythritol lipids (mel) 64 4.3. GREEN CHEMISTRY ALLIES: BIOSURFACTANTS 65 4.3.1. TOXICITY 66 5. Nanoscale zero-valent iron in remediation 67 5.1. FE TOXICITY 70 6. Biosurfactants and nanoscale zero-valent iron: state of art 71 7. Future research needs 72 8. Conclusion 73 9. References 74 3. EXPERIMENTAL DESIGN TO IMPROVE CRUDE GLYCEROL CONSUMPTION AND RHAMNOLIPIDS PRODUCTION 91 Abstract 91 1. Introduction 91 2. Materials and methods 93 2.1. MATERIALS 93 2.2. RAW MATERIAL 93 2.3. MICROORGANISMS 93 2.4. MEDIA 93 2.5. PREPARATION OF PRODUCTION MEDIUM 94 2.6. SAMPLE AND PROCESSING 94 2.7. EXPERIMENTAL DESIGN THROUGH CENTRAL COMPOSITE ROTATABLE DESIGN (CCRD) 94 2.8. STATISTICAL ANALYSIS 95 2.9. VERIFICATION EXPERIMENTS 95 3. Results and discussion 95 3.1. CENTRAL COMPOSITE ROTATABLE DESIGN 95 3.2. REGRESSION MODEL FOR RHAMNOLIPIDS (Y1) 96 3.3. REGRESSION MODEL FOR INITIAL GLYCEROL (Y2) 98 3.4. SURFACE RESPONSE METHOD (RSM) FOR RHAMNOLIPIDS (X1) AND INITIAL GLYCEROL (X2) CONCENTRATIONS 99 3.5. VALIDATION TESTS 101 4. Conclusion 102 References 103 4. RHAMNOLIPIDS AS GREEN STABILIZER OF NZVI AND ITS APPLICATION ON NITRATE REMOVAL IN SIMULATED GROUNDWATER 106 ABSTRACT 106 1. Introduction 106 2. Materials and methods 108 2.1. MATERIALS 108 2.1. PRODUCTION AND EXTRACTION OF RHAMNOLIPIDS 108 2.2. SURFACE ACTIVITY MEASUREMENTS AND STRUCTURAL CHARACTERIZATION OF RHAMNOLIPIDS 109 2.3. SYNTHESIS AND GREEN STABILIZATION OF NZVI 109 2.4. NANOPARTICLE CHARACTERIZATION 110 2.5. NITRATE REDUCTION TESTS 110 3. Results and discussion 111 3.1. RHAMNOLIPIDS PRODUCTION AND CHARACTERIZATION 111 3.2. NZVI CHARACTERIZATION 112 3.3. NITRATE REDUCTION BY NZVI IN LOW PH CONDITIONS 119 4. Conclusion 121 References 122 General conclusions 128 A. ANNEX 129 1. Selection of the Pseudomonas aeruginosa strain 129 1.1. MATERIALS AND METHODS 129 1.1.1. MICROORGANISM 129 1.1.2. GROWING CONDITIONS 129 1.1.2.1. Preparation of the inoculum 129 1.1.2.2. Flask experiments 129 1.1.3. EXPERIMENTS 130 2. Results and discussion 130 3. Conclusion 134 References 134 13 1. Introduction Nanotechnology is the production and application of extremely small structures, at the level of atoms, molecules, and supramolecular structures, whose dimensions are in the range of 1 to 100 nm (ISO, 2009; NSET, 2003). Is an innovative alternative that can be used for the remediation of contaminated sites, it has the potential to significantly affect environmental protection, because it has the property to remove the finest contaminants from water supplies and air and mitigate pollutants in the environment (NSET, 2003). Environmental pollution by organic contaminants is a major problem today. Recently, there have been many reports of soil and surface water locations contaminated with organic pollutants (CETESB, 2016; EPA, 2002; EUROPEAN COMMISSION DG; ALERT; SERVICE, 2013), with a great impact on soil and groundwater. Nanoscale zero-valent iron (nZVI) represents a new generation of environmental remediation technologies, because nZVI is non-toxic, abundant, cheap, easy to produce, and its reduction process requires little maintenance (FU; DIONYSIOU; LIU, 2014). To reduce problems with aggregation, nZVI is often coated with surfactants (CRANE; SCOTT, 2012; NSET, 2003). Surfactants play major roles improving the particle mobility (DUTRA, 2015) and lowering the interfacial tension, they also prevent coalescence of newly formed drops (MORSY, 2014). Most of the commercially available surfactants are chemically synthetized , produced based on petrochemical sources (BANAT; MAKKAR; CAMEOTRA, 2000; GAUTAM; TYAGI, 2006; VAN BOGAERT et al., 2007). Furthermore, some of the surfactants are only slowly or partially biodegradable (KUMAR, 2019) contributing to environmental impact (HAUSMANN; SYLDATK, 2015). Thus, the rapid advances in biotechnology and increased environmental awareness, the chemically surfactants have increasingly been replaced by biologically synthetized surfactants (BANAT; MAKKAR; CAMEOTRA, 2000; GAUTAM; TYAGI, 2006). Surfactants present low toxicity to humans but can affect plants and animals, i.e. ethoxylated alcohols, found in laundry detergents are toxic to fish (MULLIGAN; YONG; GIBBS, 2001a). Biosurfactants are an alternative, they can potentially replace the synthetic surfactant (REIS et al., 2013; SÁENZ-MARTA et al., 2015). Biosurfactants can be applied in bioremediation field, to clean the contaminated soil and water (THAVASI, 2011), they present advantages in microbial enhance oil recovery (MEOR) , when 14 compared to the synthetic surfactants (BANAT et al., 2010). They have low toxicity and high biodegradability and biocompatibility, additionally, present functionality under extreme conditions of temperature, pH, salinity; with possibility of production from renewable sources (BANAT et al., 2010; COOPER, 1986; DESAI; BANAT, 1997a) and the in situ application (WANG et al., 2016) Pseudomonas aeruginosa has the ability to synthesize a glycolipid-type biosurfactant, rhamnolipids this was first reported in 1949 by Jarvis et al., appud (MAIER; SOBERÓN-CHÁVEZ, 2000). It can produce rhamnolipids from substrates including C11 and C12 alkanes, succinate, pyruvate, citrate, fructose, glycerol, olive oil, glucose and mannitol (ROBERT et al., 1989). They have been intensively investigated and extensively reviewed (MAIER; SOBERÓN-CHÁVEZ, 2000; NITSCHKE; COSTA; CONTIERO, 2005; OCHSNER; HEMBACH; FIECHTER, 1996). The application of biosurfactants in the bioremediation has become one of the methods used in the remediation of contaminated sites. They can be used to clean the contaminated soil and water (THAVASI, 2011). The use of a raw material of agro-based wastes, low-cost renewable substrates and the new research about rhamnolipids applications on biodegradation and toxicity are worth further investigation and may make biosurfactants a versatile sustainable molecule. The industrial conversion of renewable resources into useful compounds has been receiving much attention; the use of crude glycerol is becoming very important from the environmental point of view (MORITA et al.; 2007; EASTERLING et al., 2009). It is a widely used substrate for rhamnolipid production (SYLDATK et al. 1985) since a wide variety of microorganisms, as Pseudomonas aeruginosa, can utilize glycerol as a source of carbon and it is often formed as an intermediate in both the aerobic and anaerobic catabolism of lipids and glucose. (JOHNSON, TACONI, 2007; HAUSMANN; SYLDATK, 2015). The hypothesis of this work lies in the fact that the rhamnolipids produced by Pseudomonas aeruginosa strain, using glycerol as a substrate may significantly increase the stabilization of nanoparticles of zero valent iron. It can become a novel application for biosurfactants allied to a consequently reduction on the costs of the process due to the use of a renewable substrate. 15 1.1. Nanotechnology The first use of nanotechnology was recorded near 600 BC in wootz steel manufacturing (WARD, 2008). The modern concept is presented in a lecture from physicist Richard Feynman in 1959 (GRIBBIN; GRIBBIN, 1997) who described a process in which the ability to manipulate individual atoms and molecules might be developed. Using one set of precise tools to build and operate another proportionally smaller set, and so on until reaching to the needed scale. 1.1.1. Nanotechnology Aplications Nanotechnology applied in biotechnology is used for biomedicine imaging and diagnostics, targeted drug delivery, nano-enabled therapies, and tissue engineering (GUPTA, 2011). Nanotechnology has been provided the foundations of drug targeting strategies. The targeted drug delivery is superior to traditional with respect to controlled release. (DEVADASU; BHARDWAJ; KUMAR, 2013; KUMARI; YADAV; YADAV, 2010). Agricultural nanotechnology can be used in plant–pathogen interactions providing new ways for crops protection. Provides an efficient management and formulations of potential insecticides and pesticides, decreasing the levels of active hazardous ingredients used in agrochemical products and applications (SRILATH, 2011). It would be useful for the development of new insect resistant varieties (RAI; INGLE, 2012). In the food sector there are a few promising applications, including nanopackaging materials that possess gas barriers and antimicrobial properties, and nanosensors which can detect microorganisms or chemical contaminants at low levels. (DUNCAN, 2011; RASHIDI; KHOSRAVI-DARANI, 2011). Also nanotechnology may develop devices for identification of nutrients deficiencies (RASHIDI; KHOSRAVI-DARANI, 2011). In civil construction the nanotechnology has focused on the structure of cement- based materials. Concrete can be nanoengineered by the incorporation of nanoparticles and nanotubes in building blocks to control material behavior and add novel properties, which can be adjusted to promote specific interfacial interactions (SANCHEZ; SOBOLEV, 2010). The compressive and flexural strength of the cement mortars nanoparticles was even higher than the strength of mortars with silica fume (LI et al., 2004; SOBOLEV; GUTIÉRREZ, 2005). Nanoparticles can act as nanoreinforcement for 16 cement phases, densifying the microstructure and the interfacial transition zone leading to a reduced porosity (SANCHEZ; SOBOLEV, 2010). Nanotechnology contributes with environmental protection, providing rapid or cost-effective clean-up on water and air as well, detecting, preventing and removing environmental contaminants (EPA, 2007; NNI, 2016; NSET, 2003). Development of nanotechnology has led to the design of cleaner industrial processes and the creation new environmentally responsible products (EPA, 2007; NSET, 2003). Due to their exceptional size, different physical and chemical properties, these nanoparticles are enabled to be utilized for innovative purposes, including the potential use in remediation (BARDOS et al., 2014; COMMISSION, 2007). 1.1.2. Types and Applications of Nanoparticles: Metallic nanoparticles exhibit size and shape-dependent properties that are of interest for applications, below there are some of the nanoparticles avaiable: Gold nanoparticles (AuNPs) They have advantages over other metal nanoparticles due to their biocompatibility, non-cytotoxicity (TOMAR; GARG, 2013). Their unique properties for imaging and therapeutic applications are used not only functionalities for specific drug delivery and cellular in bio sensing drugs but also in drug, gene and protein uptake. Doxorubicin, an anticancer drug can conjugate delivery (DAS et al., 2011; NIKAM A.P., MUKESH P., R., 2014). Silver nanoparticles (AgNPs) They have proved to be most effective because of its good antimicrobial efficacy against bacteria, viruses and other eukaryotic microorganisms (TRAN; NGUYEN; LE, 2013) food storage, textile by incorporating it into the fiber and a number of environmental applications (ABOU EL-NOUR et al., 2010; TRAN; NGUYEN; LE, 2013). Despite of decades of use, the evidence of toxicity of silver is still not clear (ABOU EL-NOUR et al., 2010). Zinc Oxide Nanoparticle (ZnONPs) It is used in vast area of applications, such as ultraviolet (UV) lasers, light- emitting diodes, photoelectrodes for the fabrication of dye-sensitized solar cells (DSSCs), antibacterial activity when with activated carbons (UMAR, 2009; VASEEM; UMAR; HAHN, 2010; YAMAMOTO; SAWAI; SASAMOTO, 2002). 17 Iron oxide nanoparticles (Fe3O4) They have reached a wide range of applications in nanotechnology, especially in the medical field, since it is chemical stability under physiological conditions and presents low toxicity (FILIPPOUSI et al., 2014), being characterized as an appropriate material for use as a magnetic resonance (MR) contrast medium, as drug delivery in specific places in the body, etc (BERGAMINI et al., 2010). 1.1.3. Physicochemical of nanotechnology applied to the stabilization process One of the major classes of materials of strategic interest is magnetic materials, owing to the different physical phenomena that are found they have become increasingly relevant to technological development (VALENTIM, 2014). The magnetic properties of nanometric particles show great differences when compared to bulky materials, they are strongly dependent on their size (RODUNER, 2006; SOUZA JUNIOR, 2012). These new characteristics come mainly from the small number of atoms per particle, when the particle size is reduced, the surface-volume relation becomes larger and the magnetic characteristics are strongly affected due to the influence of the thermal energy on the ordering of the magnetic moment, originating the phenomenon of superparamagnetism (RODUNER, 2006; SOARES JÚNIOR, 2012; SOUZA JUNIOR, 2012). 1.1.3.1. Superparamagnectic Nanoparticles The properties of the superparamagnetic nanoparticles are linked to their size. Thus, the control of the size and shape of the particles is important for the type of application. The more uniform and close the spherical shape, the greater its efficiency (COSTA, 2013). By decreasing the size of these materials to a certain critical volume − under the critical diameter (Dc)− the energy cost of creating magnetic multidomains is greater than sustaining a single magnetic domain, or monodomain state (FERREIRA, 2009; SOUZA JUNIOR, 2012; VALENTIM, 2014). When this occurs, each nanoparticle acts as a magnetic monodomain, they can present superparamagnetism (BINI, 2016; COSTA, 2013). In the Superparamagnetism the magnetic moments are randomly oriented with no net or global macroscopic magnetization, but the application of an external magnetic field causes the dipoles to align in the direction of the field. As a consequence, the field of 18 induction is added to the applied field making these materials positive magnetic susceptibility values, small and dependent on temperature (EARNSHAW, 1968). 1.2. Nanoscale Zero-Valent Iron (nZVI) The nZVI is composed of a core, which consists primarily of zero-valent iron while the mixed valent oxide shell is formed as a result of oxidation of the metallic iron (CRANE; SCOTT, 2014; LI; ELLIOTT; ZHANG, 2006). It has been found more reactive than conventional iron powder because of the large surface areas and high surface reactivity (E0 = - 0.44 V) (WEI-XIAN ZHANG, 2003; ZHAO et al., 2016). This reactivity value shows that the Fe0 is a strong reducing agent when compared to hydrogen, carbonates, sulphates, nitrates, oxygen and many organic compounds, and it can adsorb contaminants (CRANE et al., 2011; WEI et al., 2012) and for this, they have been widely investigated on the environmental remediation field (WEI-XIAN ZHANG, 2003; ZHAO et al., 2016). 1.3. Synthesis of non-stabilized nZVI particles The synthesis of nanoparticles can be made using two different strategies: the top- down and bottom-up approaches (YAN et al., 2013; ZHAO et al., 2016) (Figure 1.1). 1.3.1. Top-down approaches In the first method the nanoparticles start with a large size and the desired size is achieved by ball-milling commercial iron powder (LI; ELLIOTT; ZHANG, 2006). Li et al., (2009) have produced nZVI equivalent to a sphere with diameter of 20 nm, using no hazardous materials and producing no wastes in the synthesis process. 19 Figure 1.1 Illustration of the top-down and bottom-up approaches Source: SANCHEZ; SOBOLEV, 2010 1.3.2. Bottom-up approaches 1.3.2.1. Chemical Reduction In the other hand, with the second approach the nZVI quantity is achieved using simple chemical reagents and minimal specialist equipment. The borohydride reduction of ferrous salts or ferric ions in aqueous solution under inert conditions per reactions is the most widely studied method (WANG; ZHANG, 1997; ZHAO et al., 2016). Fe(H2O)6 3++3BH‒ 4 +3H2O→ Fe0(s) + 3B(OH)3+10,5H2(g) (1.1) 2Fe2+ + BH- + 4H2O→2Fe0(s) +B(OH) ‒ 4 + 4H+ 2H2(g) (1.2) 1.3.2.2. Carbothermal reduction The carbothermal reduction of ferrous iron has been investigated as a potential method for the manufacture of cheap and functional nZVI using thermal energy and 20 gaseous reducing agents − H2, CO2, CO, etc (Crane & Scott, 2012). The reducing gases applied are CO2 or CO produced during the thermal composition of carbon based material e.g. ultra-fine graphite powder (UG), hollow carbon nanoparticles (CNs) and carbon black (CB), using α-Fe2O3 nanoparticles (BYSTRZEJEWSKI, 2011) or aqueous solutions of iron(II) or iron(III) salts (HOCH et al., 2008). Nurmi et al., (2005) have also synthesized nZVI by reduction of goethite and hematite particles with H2 at high temperatures (200- 600 °C). Such reactions can proceed according to the following equations (1.3-1.6) (HOCH et al., 2008). Fe2(C2O4)3 ∙6H2O →2Fe + 6CO2 + 6H2O (1.3) 2Fe(C6H5O7) ∙3H2O + C → 2Fe + 2C3H6O + 6CO2 + CO + 5H2O (1.4) Fe(C2H3O2)2 + C → Fe + 2CH2CO + CO + H2O (1.5) Fe3O4 + 2C → 3Fe + 2CO2 (1.6) 1.3.2.3. Electrochemical method Another technique to produce nZVI where iron sequesters solutions ions , the product is gradually deposited on the cathode, but they often display a strong tendency towards aggregation and the formation of clusters (CRANE; SCOTT, 2012; STEFANIUK; OLESZCZUK; OK, 2016). Chen et al., (2004) have used this method with ultrasonic method and Wang et al., (2008) have used combined ion-exchange with film nafion coated on the electrode, both methods intent to disperse the nanoparticles and avoid agglomeration. The iron particle is plated on the cathode by putting ferric chloride in the solution to reduce the ferric ion to iron particle according to the following equation. Cathode: Fe3+ + 3e- + stabilizer → Fenanoparticle 1.3.2.4. Pulsed Plasma: A method in which iron nanoparticles are formed from a pulse plasma is easily controlled, yield a product is cheap and environmental safe (CHOU; PHILLIPS, 1992; KELGENBAEVA et al., 2014; YU-TAO et al., 2010). Kelgenbaeva et al. (2014) synthesized pure Fe nanoparticles with ≤10 nm size using pulsed plasma in liquid, using water–toluene interface as a medium. Yu-tao et al. (2010), presented a method based on 21 the dissociation of ferrocene using a cold argon plasma jet, the size of iron nanoparticles is about 10–30 nm for the gas phase, and 30–100 nm for the liquid phase. 1.3.2.5. Sonochemical Method Ultrasonic wave is the frequency of sound beyond the range of human hearing, above this more acoustic energy can be conveyed by the sound waves. The ultrasound waves radiate making millions of bubbles, once they reach a critical size and can no longer adsorb wave sound energy to sustain it, the surrounding liquid rushes in and the cavity violently implodes, generating microjets and shockwaves that lead to pressures reaching various atmospheres. This energy is transferred to the reaction through the solution generating heat and pressure, its known as acoustic cavitation and can accelerate the rate of chemical reactions (GATES; MAYERS; GROSSMAN, 2002; HUBER, 2005; ROOZBEH; REZA; ANVARIPOUR, 2013; TAVAKOLI; SALAVATI-NIASARI; MOHANDES, 2013). Suslick et al. (1996) introduced the sonochemical methodology, sonicating iron carbonyl in the presence of a polyvinylpyrolidone producing superparamagnetic iron nanoparticles in the range of 3 to 8 nm. Koltypin et al. (2004) prepared an airstable iron, which was exposed for two months and have not changed, using sonochemical method in the presence of edible vegetable oils. Roozbeh et al. (2013), have produced nZVI particles, their morphology changed from spherical to plate and needle type, increasing the surface area and consequently they activity. Taha et al. (2014), produced nZVI with ultrasound method, the particles were dispersed homogenously in the solution at higher speed, and rapid coalition had increased their chemical reactivity hence more Fe2+ was produced. 1.3.2.6. Biosynthesis of nZVI Living organisms including bacteria, fungi, actinomycetes and yeast have huge potential of producing intracellularly or extracellularly nanoparticles for wide applications. Many have been found to be capable synthesizing nanoparticles. By using the organisms in the reaction mixture, the production of nanoparticles with desired shape and size can be obtained (MOHANPURIA; RANA; YADAV, 2008; ZHANG et al., 2011). Njagi et al. (2011) synthesized nZVI using a green biosynthetic method employing aqueous sorghum extracts, were obtained amorphous iron nanoparticles with an average 22 diameter of 50 nm, they were tested against bromothymol blue, the results indicate that higher iron concentrations accelerated its degradation. Soliemanzadeh et al. (2016) reports the biosynthesis of nanoscale zero-valent iron (nZVI) using the extracts of Shirazi thyme leaf and pistachio green hulls, the results suggested that the synthesised nZVI with the extracts could be employed as efficient sorbents for the remediation of phosphorus. Kiruba Daniel et al., (2013) reported a nZVI biosynthesis using leaf extract of Dodonaea viscosa, the nanoparticles showed spherical morphology with an average size of 27nm, and good antimicrobial activity against Escherichia coli, Klebsiella pneumonia, Pseudomonas fluorescens and Staphylococcus aureus and Bacillus subtilis. 1.4. Surfactants Surfactants constitute an important class of industrial chemicals widely used in almost every sector of modern industry. They are amphiphilic molecules with both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases with different degrees of polarity and hydrogen bonding such as oil/water or air/water interfaces (BANAT; MAKKAR; CAMEOTRA, 2000). These properties render surfactants capable of reducing the interfacial tension, reducing the free energy of the system by replacing the bulk molecules of higher energy at an interface. Surfactants have been used in an extremely wide variety of industrial processes as adhesives, flocculating, wetting and foaming detergency, emulsification and penetrants (BANAT; MAKKAR; CAMEOTRA, 2000; GAUTAM; TYAGI, 2006; MULLIGAN; GIBBS, 1993). Most of the comercially available surfactants are chemical surfactants, produced based on petrochemical sources (BANAT; MAKKAR; CAMEOTRA, 2000; GAUTAM; TYAGI, 2006; VAN BOGAERT et al., 2007). Furthemore, some of the surfactants are only slowly or partially biodegradable contributing to environmental impact (HAUSMANN; SYLDATK, 2015). Thus the rapid advances in biotechnology and increased environmental awareness, the chemically surfactants have increasingly been replaced by biotechnologically based compounds either enzymatic or microbial synthesis (BANAT; MAKKAR; CAMEOTRA, 2000; GAUTAM; TYAGI, 2006). New legislation has provided further impetus for serious consideration of biological surfactants as possible alternatives to existing products (BANAT; MAKKAR; CAMEOTRA, 2000). 23 1.5. Biosurfactant The biosurfactant classification, is used for either surfactants derived from renewable resources with surfactant-like properties produced by microorganisms (HENKEL et al., 2012). Biosurfactants can potentially replace virtually any synthetic surfactant, they attracted attention as hydrocarbon-dissolving agents in the late 1960s (REIS et al., 2013; SÁENZ-MARTA et al., 2015). Microbial biosurfactants are extracellular compounds produced by microbes such as by bacteria, yeast, and filamentous fungi grown on different carbon sources containing hydrophobic/hydrophilic substrates (LOVAGLIO et al., 2015; NITSCHKE et al., 2005; PIRÔLLO et al., 2008a; THAVASI, 2011). The production of biosurfactants can be growth associated, in this case, they can either use the emulsification of the substrate or facilitate the passage of the substrate through the membrane. However, they are also produced from carbo-hydrates, which are very soluble. They are usually secondary metabolites, produced during the late logarithm and/ or stationary growth phases (Figure 1.2) (MULLIGAN; YONG, 2004; RON; ROSENBERG, 2001). Figure 1.2 Typical production of biosurfactants during growth Source: MULLIGAN, GIBBS, 2004 Biosurfactants are surface active molecules having hydrophilic and hydrophobic moieties as their constituents which allow them to interact at interfaces and reduce the surface tension (FERNANDES, 2011; MISHRA et al., 2009; THAVASI, 2011). All biosurfactants structure comprise a hydrophilic moiety consisting of amino acids or 24 peptides anions or cations; mono-, di-, or polysaccharides; and a hydrophobic moiety consisting of unsaturated, saturated, or fatty acids (BANAT; MAKKAR; CAMEOTRA, 2000; DESAI; BANAT, 1997b; MISHRA et al., 2009). The molecular structure often also contains several hydrophobic and corresponding hydrophilic parts. The hydrophobic part usually comprises on long-chain saturated or unsaturated fatty acids, hydroxy fatty acids or a-alkyl-b-hydroxy fatty acids, with various other structures such as isoprenoids being possible as well (HAUSMANN; SYLDATK, 2015; MULLIGAN; YONG, 2004). The hydrophilic portion can be a carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic acid or alcohol (MULLIGAN; YONG, 2004). The micellization, micelle formation is the process of spontaneous formation of aggregates in aqueous solution from a certain concentration, called the critical micelle concentration (CMC). It is an intrinsic property and characteristic of the surfactant and corresponds to the lowest amount required for increased reduction in surface tension. The micelles of the training system take place in a short interval, and is detected by the rapid changes produced in certain physicochemical properties such as surface tension, osmotic pressure and conductivity (Figure 1.3) (MANIASSO, 2001). Figure 1.3 The relationship between biosurfactant concentration, surface tension, and formation of micelles. Source: PACWA-PŁOCINICZAK et al., 2011 25 The biosurfactants have a wide variety of chemical structures and surface properties, they can be of high or low molecular weight, thus, a different group of biosurfactants have different natural growth in the producing microorganisms (Table 1.1). Therefore, one group would have advantage in a specific ecological niche and another group would be more effective in a different niche. (MULLIGAN; GIBBS, 2004; RON; ROSENBERG, 2001). Table 1.1 Type and microbial origin of biosurfactants Surfactant class Microorganism Trehalose lipids Arthrobacter paraffineus, Corynebacterium spp., Mycobacterium spp., Modococus erythropolis Rhamnolipids Pseudomonas aeruginosa, Pseudomonas sp. Sophorose lipids Candida apícola, Candida bombicola Candida lipolytica, Candida bogoriensis Glucose-, fructose-, saccharose lipids Arthrobacter sp., Corynebacterium sp., R. erythropolis Cellobiose lipids Ustilago maydis Polyol lipids Rhodotorula glutinus, Rhodotorula graminus Diglycosyl diglycerides Lactobacillus fermentii Lipopolysaccharides Acinetobacter calcoaceticus (RAGl), Pseudomonas sp. Candida lipolytica Lipopeptides Arthrobacter sp., Bacillus pumilis, Bacillus licheniformis Surfactin Bacillus subtilis Viscosin Pseudomonas fiuorescens Ornithine, lysine peptides Thiobacillus thiooxidans, Streptomyces sioyaensis Gluconobacter cerinus Phospholipids Acinetobacter sp. Sulfonylipids T. thiooxidans, Corynebacterium aUamolyticum Fatty acids (corynomycolic acids, Capnocytophaga sp., Penicillium spiculisporum spiculisporic acids, etc.) Capnocytophaga sp., Penicillium spiculisporum Corynebacterium lepus, Arthrobacter paraffineus Talaramyces trachyspermus, Nocardia .erythropolis Source: Mulligan; Gibbs 1993 High molecular weight polymeric biosurfactant: Are produced by many bacteria of different species, such as various prokaryotes, including Archaea, Gram-positive, and Gram-negative bacteria. They are polysaccharides, proteins, lipopolysaccharides, lipoproteins, or complex mixtures of these biopolymers; complex mixtures of these referred to as lipoheteropolysaccharides (HAUSMANN; SYLDATK, 2015; RON; ROSENBERG, 2001). Polymeric biosurfactants has properties like high viscosity, high tensile strength, and resistance to shear. It is because of these properties that they have found a variety of industrial uses in pharmaceuticals, cosmetics, and food industries (HAUSMANN; SYLDATK, 2015). Low molecular weight: based on their composition, they can be glycolipids or lipopeptides, but may also belong to this group simple fatty acids and free phospholipids 26 (MAKKAR; CAMEOTRA, 2002; PIRÔLLO et al., 2008b; SÁENZ-MARTA et al., 2015). The lipopeptides are most known for their antibiotic properties, among then the Bacillus subtilis produces a cyclic lipopeptide called surfactin, its amphipactic nature may contribute to the formation of ion-conducting pores in membrane (GRAU et al., 1999; PEYPOUX; BONMATIN; WALLACH, 1999). Free fatty acids or phospholipids can be form by some bacteria and fungi when growing on n-alkanes (DESAI; BANAT, 1997). Fatty acids are components of a long-chain aliphatic carboxylic acid found in natural fats and oils, they also constitute membrane phospholipids and glycolipid Phospholipids are lipids containing one or more phosphate groups, are the main part of microbial membranes and are usually not present in an extracellular form (HAUSMANN; SYLDATK, 2015). Within the biosurfactants, the glycolipids form the greatest share, with the non- sugar component, the aglycone, being highly versatile. These structures are particularly interesting since many biosurfactants exhibit high biological degradability (HAUSMANN; SYLDATK, 2015; MULLIGAN; YONG, 2004). They are carbohydrates in combination with long-chain aliphatic acids or hydroxyaliphatic acids (GAUTAM; TYAGI, 2006). Glycolipids comprising mono or oligosaccharides as well as lipid moieties form the most important group of low molecular weight biosurfactants. Biosurfactants with low molecular mass are efficient in lowering surface and interfacial tensions, whereas biosurfactants with high molecular mass are more effective at stabilizing oil-in-water emulsion. (BANAT; MAKKAR; CAMEOTRA, 2000; HAUSMANN; SYLDATK, 2015). Among the glycolipids, the four important groups of microbial glycolipids are rhamnolipids, sophorolipids, trehaloselipids, and mannosylerytitollipids. (BANAT; MAKKAR; CAMEOTRA, 2000; DESAI; BANAT, 1997b; HAUSMANN; SYLDATK, 2015; RON; ROSENBERG, 2001). 1.6. Production of Rhamnolipids Pseudomonas aeruginosa is defined as a Gram negative bacterium non-sporulated, can be found and isolated from different habitats, such as soils and plants, wetlands, marine and coastal habitats and animal tissue (OLIVEIRA, 2010). P. aeruginosa was first reported in 1949 by Jarvis and associates (MAIER; SOBERÓN-CHÁVEZ, 2000). It has the ability to synthesize a lot of glycolipid, type rhamnolipids. Pseudomonas aeruginosa can produce rhamnolipids from substrates including C11 and C12 alkanes, succinate, 27 pyruvate, citrate, fructose, glycerol, olive oil, glucose mannitol, oil, fats fatty acid, and lignocellulose (HENKEL et al., 2012; ROBERT et al., 1989). Rhamnolipids have been intensively investigated and extensively reviewed (MAIER; SOBERÓN-CHÁVEZ, 2000; NITSCHKE et al., 2005; OCHSNER; HEMBACH; FIECHTER, 1996) (Table 1.2). They are capable of reducing the surface tension of water from 72 mN/m to values between 25 and 30 mN/m (APARNA; SRINIKETHAN; HEGDE, 2011). Table 1.2 P. aeruginosa strain in various carbon source. Strain Carbon Source BIBLIOGRAPHY DSM2659 Glucose GUERRA-SANTOS ET AL., 1984 UG2 and PG201 Naphthalene DEZIEL ET AL., 1996 * Crude Oil ZHANG ET AL., 2005 LBI Soybean, Corn, Babassu, Cottonseed, and Palm Oil NITSCHKE ET AL., 2005B P029-GVIIA Molasses OLIVEIRA, 2010 LBI Benzene, Toluene, Diesel Oil, Crude Oil and Oil Sludge PIRÔLLO ET AL., 2008 GS9-119 and DS10- 129 safflower oil, soybean oil, or glycerol RAHMAN ET AL., 2002 44T1 Dodecane ROBERT ET AL., 1989 LBI Oils from Buriti, Cupuaçu, Passion Fruit, Andiroba, Brazilian Nut and Babassu COSTA ET AL., 2006 L2-1, B1-3, 6c, and 7a Cassava Wastewater, Waste Cooking Oil and Glycerol COSTA ET AL., 2009 DSM 2874 Rapeseed Oil TRUMMLER ET AL., 2003 PAO1 Sunflower Oil MÜLLER ET AL., 2010 OG1 Waste Frying Oil and Ram Horn Peptone ÖZDAL ET AL., 2017 LBI Soapstock BENINCASA ET AL., 2002 KY 4025 n-Paraffin ITOH ET AL., 1971 47T2 Frying Oil HABA ET AL., 2000 J4 Glycerol, Glucose, Grape Seed Oil, Olive Oil and Sunflower Oil WEI ET AL., 2005 PG201 Glucose OCHSNER ET AL., 1995 DSM 2659 Glucose and Hexadecane KOCH ET AL., 1991 S7B1 n-hexadecane or n-paraffin HISATSUKA ET AL., 1971 AT10 Casablanca crude oil ABALOS ET AL., 2004 *Note: the strain of P. aeruginosa has not been presented by the authors. Rhamnolipids comprise one or two molecules of rhamnose are linked to one or two molecules of b-hydroxydecanoic acid (BANAT; MAKKAR; CAMEOTRA, 2000; DESAI; BANAT, 1997a; HAUSMANN; SYLDATK, 2014). Consist mainly of a mono and di-rhamnolipids mixing and linked to one or two β acid molecules - reported hydroxydecanoic as RL1 (Rha2C10C10), RL2 (RhaC10C10), RL3 (Rha2C10) and RL4 (RhaC10) Figure 1.4. 28 Figure 1.4 Structure of the four main counterparts of rhamnolipids Source: LOVAGLIO, 2011 The type and proportion of produced rhamnolipids depend on the strain, on the carbon source used concentration of the medium components, pH, nutrient composition, substrate and temperature (DESAI; BANAT, 1997b; GAUTAM; TYAGI, 2006; GUERRA-SANTOS; KAPPELI; FIECHTER, 1984; LOVAGLIO et al., 2014; MULLIGAN; GIBBS, 1993). Temperature can cause alteration in the composition of the biosurfactant produced. (SYLDATK et al., 1985), according to Mulligan; Gibbs (1993), rhamnolipid production by P. aeruginosa is optimal at pH 6.25 and 33°C; at higher pH (> 7.5), production ceased. Rhamnolipid production reaches its maximum at a pH range 6 - 6.5 and decreased sharply above pH 7 (GUERRA-SANTOS, 1984). Depending on the number and length of β-hydroxyl fatty acid chains and the number of rhamnose residues, the critical micelle concentrations (CMC) of rhamnolipids can vary from 5 to 200 mg/l (NITSCHKE; COSTA; CONTIERO, 2005). Because of their unique structures, biosurfactants may have a large range of properties that can be exploited commercially (SÁENZ-MARTA et al., 2015). Considering the important properties and a wide range of applications of biosurfactants, much more attention has been given to understand the biochemical properties and 29 physiological role on the producing microorganism as well as commercial application of biosurfactants (WARD, 2010). Even though microbial surfactants possess diverse structures and better chemical properties than the synthetic equivalents, they could not overcome the chemical surfactants in cost and production capacity (HAUSMANN; SYLDATK, 2015). Successful production depends on development of cheaper processes and the use of renewable materials and low cost (MAKKAR; CAMEOTRA, 1999) Reduction of production costs is, therefore, necessary to establish microbial surfactants as a general alternative. The high cost of production due to the use of expensive substrates which account for the 10–30% of the overall costs and inefficient methods, have prevented its widespread use (HEYD et al., 2008; PARRA et al., 1989). 1.7. Process of bioremediation Bioremediation can be defined as an engineered technology that modifies environmental conditions chemical, physical or biochemical, such as pH, moisture and aeration to encourage microorganisms to degrade hazardous organic constituents to harmless substances (EPA, 2013; WILSON; JONES, 1993). The process can be applied ex situ if the contaminant is carried away from the contaminated site to a treatment facility in tanks, biopiles, or other treatment systems or in situ if the contaminant is treated at the contaminated site (EPA, 2013; MACAULAY; REES, 2014). It uses naturally occurring bacteria and fungi or plants to degrade complex chemical compounds. (MACAULAY; REES, 2014; SINGH; SINGH; SHARMA, 2014). There are many toxic materials generated as byproducts from several industries, which may be released into the environment. Among the heavy metals, the most hazardous are in an Environmental Protection Agency’s list of priority pollutants, which includes cadmium, copper, lead, mercury, nickel and zinc (CAMERON, 1992).The biological remediation is under development and is considered low cost, in relation to conventional processes (MULLIGAN; YONG; GIBBS, 2001a). The bioremediation of hydrocarbons occurs through increment of the solubility and bioavailability provided by the biosurfactants, their ability to enhance the surface area of hydrophobic water-insoluble substrates leads to removal of the contaminants by pseudo solubilization and emulsification (BANAT et al., 2010; RON; ROSENBERG, 2002). In the bioremediation process the biosurfactant can be added to as purified 30 materials or in the form of bioemulsifier in both situations, they can stimulate the growth of oil-degrading bacteria (RON; ROSENBERG, 2002), resulting in the degradation of these compounds and their elimination from the environment (JACQUES et al., 2007). Some studies described that the biosurfactant by Bacillus subtilis showed efficiency in removing Zn, Cu, Cr and hydrocarbons from contaminated systems (DE FRANÇA et al., 2015). Also, the biosurfactants from Bacillus subtilis was used to degradation of used motor oil from contaminated sand, the removal rage achieved 82% (BEZZA; CHIRWA, 2015). The crude biosurfactant from Serratia marcescens showed ability to emulsify petroleum derivatives and also, exhibited properties of dispersing engine oil-in-water (78%) and to remove burned engine oil in beach sand (88.27%) and mangrove sediment (73.70%). Stenotrophomonas sp. produced a biosurfactant with capacity to reduce oil spillage in both aqueous and soil phases which also exhibited antimicrobial and antioxidant properties, this could be interesting for food, cosmetics, and detergents industries (GARGOURI et al., 2017). The application of biosurfactants in the remediation of heavy metals, when added to washing water promote desorption, solubilize and dispersal of heavy metals through complexation of the free form of the metal (Banat et al., 2010; Miller, 1995; Mulligan et al., 2001). The biosurfactant from Candida sphaerica can remove Fe, Zn and Pb from contaminated soil and aqueous solution, indicating removal rates among 95% and 79% (LUNA; RUFINO; SARUBBO, 2016). Chen et al., (2017) used rhamnolipids to remove heavy metals in a river sediment and observed that it was favored at high concentration, long washing time, and high pH, the efficiency was closely related to the original speciation of heavy metals in sediment. Yang et al., (2016) reported that the glycolipid produced by Burkholderia sp was efficient in the removal of Zn, Mn, Cd from contaminated soils, this result may come from the formation of a metal complex with biosurfactant and adhesion of heavy metal and minerals with the microorganism. 1.8. Rhamnolipids in bioremediation There are many toxic materials generated as byproducts from several industries, which may be released into the environment. Aromatics and their chlorinated derivatives, which are toxic, are generated in pesticide and herbicides industries, petroleum and petrochemicals industries, plastics, iron and steel industries, wood preservation, etc 31 (KOSARIC, 2001). Among the heavy metals, the most hazardous are in an EPA’s list of priority pollutants, which includes cadmium, copper, lead, mercury, nickel and zinc (CAMERON, 1992).The biological remediation is under development and is considered low cost, in relation to conventional processes (MULLIGAN; YONG; GIBBS, 2001a). The bioremediation of hydrocarbons occurs through increased of the solubility and bioavailability provided by the biosurfactants, its ability to enhance the surface area of hydrophobic water-insoluble substrates leads to removal of the contaminants by pseudo solubilization and emulsification. (BANAT et al., 2010; RON; ROSENBERG, 2002). In the bioremediation process the biosurfactant can be added to as purified materials or in the form of bioemulsifier in both situations, they can stimulate the growth of oil-degrading bacteria (RON; ROSENBERG, 2002), resulting in the degradation of these compounds and their elimination from the environment (JACQUES et al., 2007). Santos et al., (2013) observed that the crude biosurfactants from Candida lipolytica and aqueous solutions of the isolated biosurfactantat were effective in recovering up the motor oil from the walls of the beakers. The biosurfactants produced by Pseudomonas sp and Sphingomonas sp combined with electrokinetic (EK) remediation degraded phenanthrene in the soil with an efficiency of up to 65,1 % and 49,9 % at the (LIN et al., 2016). The comparison of rhamnolipids, Tween 80, and sodium dodecyl benzenesulfonate (SDBS) sprayed onto soils contaminated with polycyclic aromatic hydrocarbons (PAHs), showed a degradation of 95 % in the soil treated with rhamnolipids, followed by 92 % degradation with Tween 80 and 90 % degradation with SDBS (WANG et al., 2016). Jain et al. (1992) observed that the biodegradation of tetradecane, hexadecene and pristane was significantly enhanced by adding either Pseudomonas aeruginosa UG2 celIs or their biosurfactants in soil with this hydrocarbon mixture. The biosurfactant from P. aeruginosa strain exhibited a higher performance in oil recovery than the chemical surfactants Enordet and Petrostep, suggesting its potential application in the oil industry or bioremediation processes (GUDIÑA et al., 2015). In an experiment simulating marine oil spill bioremediation using a bacterial consortium with rhamnolipids, the hydrocarbons biodegradation was enhanced with an efficiency by 5,6% (CHEN et al., 2013). The application of biosurfactants in the remediation of heavy metals, when added to washing water promote desorption, solubilisation and dispersal of heavy metals 32 through complexation of the free form of the metal (BANAT et al., 2010; MILLER, 1995; MULLIGAN; YONG; GIBBS, 2001b). The biosurfactant from Candida sphaerica in the removal of Fe, Zn and Pb from soil collected from an automotive battery industry and from aqueous solution indicates removal rates among 95% and 79% (LUNA; RUFINO; SARUBBO, 2016). Chen et al., (2017) analyzed the effects of rhamnolipid as the washing agent to remove heavy metals in river sediment was favored at high concentration, long washing time, and high pH., the efficiency was closely related to the original speciation of heavy metals in sediment. 1.9. Crude Glycerol Biodiesel has recently experienced a considerable production increase in the world. A rapid expansion in production capacity is being observed not only in developed countries but also in developing ones (CARRIQUIRY, 2007). Various countries have recently adopted the use of biodiesel mixture in petroleum, in Brazil, the addition of 2% biodiesel (B2) is obligatory since January of 2008, increasing to 5% (B5) in 2013 (DA SILVA; MACK; CONTIERO, 2009). Biodiesel is mainly produced by the transesterification of animal fats or vegetable oils (NANDA et al., 2014). Crude glycerol is the primary byproduct of the transesterification process in a biodiesel plant, (Figure 1.5) between oils or fats (triglycerides) and an alcohol (usually methanol). The feedstocks usually are pure vegetable oil (e.g., soybean, rapseed, sunflower), rendered animal fats, or waste vegetable oils. The theoretical ratio of methanol to triglyceride is 3:1; one methanol molecule for each of the three hydrocarbon chains and is equivalent to approximately 12% methanol by volume. (AGGELIS, 2009; ZHOU et al., 2008). 33 Figure 1.5 Transesterification reaction of a triacylglycerol with an alcohol. Source: FELTES et al, 2011 Glycerol, 1,2,3-propanetriol, is a colorless, odorless, viscous liquid with a sweet taste, derived from both natural and petrochemical feedstocks (PAGLIARO; ROSSI, 2008, 2010). Glycerol can be obtained by basic hydrolysis of triglycerides of animal fat or vegetable oil, by microbial fermentation or by chemical synthesis from petrochemical feedstocks or can be recovered as a by-product of soap manufacture from fats. In general, the conversion of glycerol can be broken down into two classes: (1) oxidation or reduction of the glycerol into other three carbon compounds, or (2) reaction of glycerol with other molecules to form new species. Glycerol is furthermore a relevant by-product in biodiesel production (HAUSMANN; SYLDATK, 2015; WANG et al., 2001). Is a simple alcohol with many uses in the cosmetic, paint, automotive, food, tobacco, pharmaceutical, pulp and paper, leather and textile industries or as a feedstock for the production of various chemicals (WANG et al., 2001). Is one of the most versatile and valuable chemical substances known, is completely soluble in water and alcohols, is slightly soluble in many common solvents such as ether and dioxane, but is insoluble in hydrocarbons. Contains three hydrophilic alcoholic hydroxyl groups, which are responsible for its solubility in water and its hygroscopic nature. It is a flexible molecule forming both intra- and intermolecular hydrogen bonds (PAGLIARO; ROSSI, 2008, 2010). The industrial conversion of renewable resources into useful compounds has been receiving much attention from the environmental point of view; the use of crude glycerol is becoming very important, because the increasing production of biodiesel 34 (EASTERLING et al., 2009; MORITA et al., 2007). Glycerol is a widely used substrate for rhamnolipid production (SYLDATK et al., 1985) since a wide variety of microorganisms can utilize glycerol as a source of carbon and it is often formed as an intermediate in both the aerobic and anaerobic catabolism of lipids and glucose (HAUSMANN; SYLDATK, 2015). The bioconversion of glycerol adds expressive value to the biodiesel industry (DA SILVA; MACK; CONTIERO, 2009). The choice of inexpensive raw materials is extremely important in the total economy of the process because they account for 50% of the final product cost. (MAKKAR; CAMEOTRA, 1999). However, are certain disadvantages of using low-priced substrates. For example, glycerol can be obtained in different purities, crude glycerol, used for the production of biodiesel, which contains approximately 90% glycerol may contain various types of impurities such as methanol, salts, soaps, heavy metals, and residual fatty acids (PAGLIARO; ROSSI, 2010; SOUSA et al., 2011). Due to the presence of such heavy impurities, its purification for industrial applications is unprofitable (CHATZIFRAGKOU et al., 2010; CHATZIFRAGKOU; PAPANIKOLAOU, 2012), nevertheless these impurities could difficult the access of the glycerol by the microorganisms. Thereby, the utilization of glycerol through biotechnological approaches represents a hopeful guarantee for the its effective management (PAPANIKOLAOU, 2009), many studies has been reported on their use for the production of biosurfactants (Table 1.3). 35 Table 1.3 Production of Rhamnolipids by Pseudomonas strains using Glycerol Substrate Average Content Strain Rhamnolipid (g/L) Reference Crude glycerol (biodiesel production) 80% DSM 2874 8.5 SYLDATK et al., 1985 Glycerol * DSM2659 1.0 SANTA ANNA et al., 2002 Glycerol * DS10-129 1.77 RAHMAN et al., 2002 Glycerol 3% PA1 6.9 SANTA ANNA et al., 2002 Glycerol 4% DSM 2874 1.77 TRUMMLER; EFFENBERGER; SYLDATK, 2003 Glycerol 3% - 15.4 ZHANG et al., 2005 Glycerol 3% UCP0992 8 SILVA et al., 2010 Glycerol 50g/L EQ 109 0.68 SANTOS, 2011 Crude glycerol (biodiesel production) 2% MSIC02 2.3 SOUSA et al., 2014 Glycerol 3% BK-AB12 0.048 PUTRI; HERTADI, 2015 Glycerol 1% WAE 2.164 ERAQI et al., 2016 Crude glycerol (biodiesel production) 50 g/L LBI 2A1 2.55 SALAZAR-BRYAM; LOVAGLIO; CONTIERO, 2017 *Note: average content not specified by the authors. 2. Background and Project Relevance The use of nanoscale zero-valent iron is a promising technology for environmental remediation by reductive degradation of organic compounds in environments low in dissolved oxygen. However, their physical and chemical properties and their reactivity are not yet fully known. Furthermore, strong aggregation of nZVI and low stability at storage conditions inhibit the dispersion in the treating zones, making the use of surfactants an alternative to stabilize nZVI. In this scenario the rhamnolipids produced by Pseudomonas aeruginosa, using glycerol as a substrate can be presented as a solution to this problem. The use of biosurfactants in the bioremediation has become one of the methods used in the remediation of contaminated sites (THAVASI, 2011). The motivation for this work is to give subsidies to the use of rhamnolipids, produced by the bacterium Pseudomonas aeruginosa, in the stabilization process and remediation using nZVI. The rhamnolipids may significantly increase stabilization of nanoparticles of iron zero valent. 36 3. Objectives General Stabilization of de nanoscale zero-valent iron (nZVI) aiming the biosurfactant produced by Pseudomonas aeruginosa using glycerol as a carbon source. Specific 1. A bibliographic survey about the state of the art of nZVI and biosurfactants; 2. Optimization of temperature and initial carbon source for the production of rhamnolipids and glycerol consumption using Pseudomonas aeruginosa LBI 2A1; 3. Production, stabilization and characterization of nZVI using rhamnolipids as capping agent; 4. Study the performance of bare nZVI and stabilized nZVI in nitrate removal. References ABALOS, A. et al. Enhanced Biodegradation of Casablanca Crude Oil by A Microbial Consortium in Presence of a Rhamnolipid Produced by ... n. April, p. 249–260, 2004. ABOU EL-NOUR, K. M. M. et al. Synthesis and applications of silver nanoparticles. 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