Universiẗat, Diss., 2000--Stuttgart.

All fossil fuels contain varying levels of sulfur compounds which are undesirable because they cause environmental pollution, corrosion, acid rain and lead to health problems. There is strict international legislation for the permissible levels of sulfur compounds in fossil fuels. The aim of this research therefore was the biocatalyst development for biodesulfurisation using two approaches. In the first approach, Rhodococcus erythropolis IGTS8-5 and IGTS8-5G were immobilised in porous coke particles and tested in repeated cycles successfully. Both bacterial strains grew well in the chemically defined medium with glucose as the main carbon and energy source and the model sulfur compound dibenzothiophene (DBT) as the sole sulfur source. 0.8 g of cells was immobilized on 250 g of coke particles without refreshing the medium over 72 h while 1.8 g of cells were immobilised on 250 g of coke when the media was refreshed every 24 hours for 120 h after the initial immobilisation batch of 72h. The latter, were used repeatedly in twelve consequtive batch desulfurisation cycles during which the biodesulfurisation activity progressively decreased from over 95% removal of 100 ppm DBT to around 45% removal. DBT removal is often expressed in terms of 2-hydroxybiphenyl which is the end product of biodesulfurisation. The biodesulfurisation activityof immobilised bacteria was equivalent to 310 umol 2-HBP h-1g-1 dry cell weight during the first hour. Freely suspended cells on the other hand exhibited biodesulfurisation activity equivalent to 91 umol 2-HBP h-1g-1 dry cell weight. Unfortunately, after the first 24 h, the activity of the immobilised cells decreased to 12 umol 2-HBP h-1g-1 dry cell weight. Use of plant cell cultures for biodesulfurisation is the other novel aspect of this work. Armoracia rusticana (horse radish) cell culture was chosen as the novel biocatalyst since this plant is a well known source of peroxidase enzyme which is involved in the biodesulfurisation metabolism according to the literature on bacterial biodesulfurisation. Arabidospsis thaliana (thale cress) was also used since its genome is completely sequenced and it is a model organism in genomics studies. Our results indicate that cell suspensions of both plants did show biodesulfurisation activity by reducing the level of sulfur compounds, mainly DBT and other three derivatives from both aqueous and oil phase. When compared to the bacteria, in terms of DBT consumption, the activity of A. rusticana was calculated as 55 umol DBT h-1 g-1 DCW and 65 umol DBT h-1 g-1 DCW for A. thaliana while in bacteria it was 91 umol DBT h-1 g-1 DCW for IGTS8-5 and 73 umol DBT h-1 g-1 DCW for IGTS8-5G. Transcriptomics analysis of the plant cell cultures after exposure to the DBT when compared to control cultures showed alterations in gene expression levels several of which were related to sulfur metabolism and transmembrane transporters of sulfate.

Arsenic (As) is a toxic metalloid and the remediation of soils and waters from this contaminant as well as the prevention of future contamination are still pending tasks in Chile. There are bacteria able to live in environments polluted with arsenic, as they have tolerance mechanisms for this metalloid, or even can use it for energy metabolism. The potential tolerance mechanisms include the production of siderophores, metabolites with chelating activity that can decrease the toxicity of metals and metalloids. Although a correlation between siderophore production and metalloid tolerance has been described, the structure of arsenic-binding siderophores and their implications in tolerance have not been elucidated yet. In this work, it is proposed that bacteria isolated from contaminated environments produce arsenic-binding siderophores. The main aims of this work are to study the production of the siderophores by arsenic-tolerant bacteria, to characterize these compounds and to determine their relation with tolerance to arsenic. Fourteen arsenic-tolerant bacteria were isolated from contaminated water, From these, four strains belonging to the species Rhodococcus erythropolis, Arthrobacter oxydans and Kocuria rosea were selected, in addition to the previously isolated Rhodococcus erythropolis S43, for a more detailed study. The isolates were used to produce siderophore extracts, which were then evaluated for their iron- and arsenic-binding activity. To detect the latter, a new method (As-mCAS) was set up, based on the Chrome Azurol S (CAS) test, an assay to detect iron-chelating activity of siderophores. After testing the extracts, R. erythropolis S43 was selected as the strain with the best arsenic-binding activity. For the subsequent chemical characterization, siderophores were produced under control conditions (iron-free M9 medium) and under stress conditions with arsenic (iron-free M9 medium with sodium arsenite). HPLC analysis of the extracts for both culture conditions showed the presence of a single compound with both an iron-chelating and an arsenic-binding activity. Analyses by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) for both culture conditions suggested the main presence of the siderophore heterobactin B. In addition, the genome of strain S43 was sequenced. A cluster of ars-genes was predicted, probably responsible for the arsenic-tolerance of the strain. In addition, a complete gene cluster for heterobactin production was found. However, no significant difference was obtained in the expression of these determinants in the presence or absence of arsenic, suggesting that the production of this siderophore in strain S43 is not responsible for the tolerance to the metalloid.

[pt] A remoção de espécies metálicas em baixas concentrações a fim de atingir os padrões exigidos pela legislação vigente, tem se mostrado um desafio. A biossorção pode ser definida como um processo físico-químico que remove substâncias de uma solução aquosa onde o sorvente é uma matriz biológica. Este é um processo reversível e de ligação rápida das espécies metálicas nos grupos funcionais presentes na superfície da biomassa. Neste trabalho foi avaliada a capacidade biossortiva da cepa bacteriana Rhodococcus erythropolis e da casca de banana modificada quimicamente na remoção de Ni2 positivo e Co2 positivo em soluções aquosas em ensaios de batelada. Foi avaliado também a flotação biossortiva utilizando a R. erythropolis devido seu alto grau de hidrofobicidade, além de agir como bioespumante e biocoletora. O valor de pH adequado para a biossorção com R. erythropolis foi entorno de 7 para ambos metais e para a casca de banana modificada como biossorvente foi entre 6 e 7 para ambos metais. Os resultados da capacidade de captação dos biossorventes foram bem ajustados ao modelo de isoterma de Langmuir, onde as capacidades de captação máximas utilizando a casaca de banana modificada foram de 29,35 mg/g para Ni2 positivo e 24,70 mg/g para Co2 positivo e utilizando a R. erythropolis foram de 14,46 mg/g para Ni2 positivo e 16,06 mg/g para Co2 positivo. As propriedades superficiais dos biossorventes foram avaliadas antes e após a interação dos metais para poder determinar os possíveis mecanismos de biossorção, mediante medições eletrocinéticas, espectroscopia de infravermelho e microscopia eletrônica de varredura. Nos ensaios de flotação biossortiva foi verificado que a R. erythropolis apresenta propriedades como biorreagente (bioespumante e biocoletora), onde foi obtido porcentagens de remoção de 40,20 e 23,52 porcento para o níquel e cobalto, respectivamente. Os resultados apresentados mostram que apesar de ambos os biossorventes se apresentarem como alternativa para remoção de metais em efluentes de baixa concentração a casca de banana modificada apresentou uma maior remoção. / [en] The removal of metallic species at low concentrations in order to reach the standards required by current legislation has been a challenge. Biosorption can be defined as a physicochemical process that removes substances from an aqueous solution where the sorbent is a biological matrix. This is a fast and reversible binding process between the metallic species and the functional groups present on the surface of the biomass. The main objective of this work was to evaluate the biosorptive capacity of the bacterial strain Rhodococcus erythropolis and banana peel chemically modified on the removal of Ni2 positive and Co2 positive in aqueous solutions in batch. Biosorptive flotation was also evaluated using R. erythropolis due to its high hydrophobicity degree. The appropriate pH value for biosorption with R. erythropolis was around 7 for both metals and for biosorbent modified banana peel was between 6 and 7 for both metals. The results of the uptake capacity of the biosorbents were well adjusted by the Langmuir isotherm model, where the maximum capture capacities using the modified banana peel were 29.35 mg/g for Ni2 positive and 24.70 mg/g for Co2 positive, and, using the Rhodococcus erythropolis bacteria were 14.46 mg/g for Ni2 positive and 16.06 mg/g for Co2 positive. The surface properties of the biosorbents were evaluated before and after interaction with the metallic species in order to determine the possible mechanisms of biosorption, through electrokinetic measurements, infrared spectroscopy and scanning electron microscopy. In the biosorptive flotation experiments it was verified that R. erythropolis presents properties as biocollector and biofrother, achieving removal percentages of 40.20 percent and 23.52 percent for nickel and cobalt, respectively. The results show that although both biosorbents present significant behavior for metal removal at low concentration the modified banana peel showed a greater removal.

The main goal of this thesis is to expertise biological treatment of sewage water rising from production of DPG (diphenylguanidine). The theoretical part is focused on the water and means of sewage disposal. There is shortly described mechanism of physical, chemical, biological and combinated methods of sewage disposal. In practical part of this thesis, focused on expertising degradation of DPG and aniline was chosen high performance liquid chromatography (HPLC) technique, which made it possible to determined their biodegradation abilities. The next component of this thesis are sideline products determination, phenylurea especially. This thesis is focused on monitoring of biofilm´s character and the activity of Rhodococcus erythropolis bacterial culture, used in biodegradation of DPG in chemical-biological wastewater treatment plant, LZD a.s.

[pt] A interação entre bioreagentes e minerais vem sendo estudada por muitos anos. Os biossurfactantes de bactérias Rhodococcus opacus e Rhodococcus erythropolis podem se comportar como coletores e espumantes na flotação mineral. Este trabalho teve como objetivo avaliar a interação de diferentes porcentagens de misturas de Rhodococcus opacus e Rhodococcus erythropolis para recuperação de hematita de um rejeito minério de ferro de baixo teor, com o quartzo como principal mineral de ganga. A interação dos dois biossurfactantes foi investigada usando diferentes técnicas como a tensão superficial, o potencial zeta, o ângulo de contato (Ɵc) e a espectroscopia no infravermelho com transformada de Fourier (FTIR) para entender o comportamento nas diferentes interfaces. As análises de tensão superficial de cada biossurfactante em contato com a água atingiram o menor valor de 32.35 mN.m-1. (Rhodococcus opacus) e 31.41 mN.m-1. (Rhodococcus erythropolis) em meio ácido pH 3. As medições experimentais do potencial zeta revelaram a interação de ambos biossurfactantes nas superfícies da hematita e do quartzo. O ponto isoelétrico (IEP) da hematita foi próximo ao pH 3.40 e o ponto de reversão de carga depois de interagir com os biossurfactates Rhodococcus opacus e Rhodococcus erythropolis foi alcançado em pH 2.20 e pH 3.30. O IEP do quartzo ficou em torno de pH 2.12 e o potencial zeta diminuiu quando os dois biossurfactantes foram adicionados, não sendo possível obter o deslocamento do IEP após a interação com eles. O FTIR confirmou a afinidade da superfície da hematita com os dois biossurfactantes, ocorre o contrário no caso do quartzo, já que houve uma fraca adsorção na superfície. Finalmente o Ɵc da hematita (40°) após a interação por separado com biosurfactante do R. opacus mudou para 64° e com o biosurfactante R. erythropolis variou para 87°, estas mudanças mostraram uma boa hidrofobização da hematita. Os resultados dos testes de microflotação na célula modificada de Partridge-Smith revelaram que o aumento da proporção percentual de Rhodococcus erythropolis levou a um aumento na recuperação metalurgia de 48.07% e um teor de concentrado de ferro de 24.35%. / [en] The interaction between different bioreagents and minerals has been studied for many years. Rhodococcus opacus and Rhodococcus erythropolis biosurfactants can behave as collectors and frothers in mineral flotation. This work aims to evaluate the interaction between them mixed in different percentages for recovering hematite from an iron ore tailing composed of low grade of hematite and quartz as gangue. The interaction of these two biosurfactants was investigated using different techniques such surface tension, zeta potential, contact angle (Ɵc), and Fourier Transform Infrared (FTIR) to understand the behaviour at the different interfaces. The surface tension analyses of each biosurfactant in contact with water reached the lowest value in 32.35 mN.m-1 (Rhodococcus opacus) and 31.41 mN.m-1 (Rhodococcus erythropolis) at acidic conditions pH 3. The experimental zeta potential measurements revealed the positive interaction of both biosurfactants on the hematite surface. The isoelectric point (IEP) of hematite was near pH 3.40 and the charge reversal point of Rhodococcus opacus and Rhodococcus erythropolis was achieved in pH 2.20 and pH 3.30 respectively. The IEP of quartz was around pH 2.12 and the zeta potential decreased when both biosurfactants were added, it was not possible to get the displacement of the IEP after interaction with them. The FTIR analyses confirmed the affinity of hematite surface with both biosurfactants, the opposite happens in the case of quartz, there was weak adsorption on the surface. Finally, the Ɵc of hematite 40 ° after interaction with R. opacus biosurfactant increased to 64° and with R. erythropolis biosurfactant amplified to 87°. This changes indicate a good hydrophobization of hemtatite. The results of microflotation tests in the Partridge-Smith modified cell revealed that a huge percent proportion of R. erythropolis led to an increase in the metallurgic recovery of about 48.07% and 24.35 % of iron concentrate.

[pt] A crescente demanda mundial por matérias-primas minerais levou ao aumento da exploração mineral e paralelamente novas pesquisas estão sendo dirigidas para a produção de novos reagentes de flotação, a fim de que estes apresentem maior seletividade e não sejam agressivos ao meio ambiente. Nesta pesquisa teve-se como objetivo estudar os aspectos fundamentais da bioflotação da hematita, avaliando a cepa bacteriana Rhodococcus erythropolis como biocoletor. Entre os estudos efetuados estão análises química para determinar as proteínas e carboidratos presentes no concentrado bacteriano, estabelecendo-se que é constituída por macromoléculas com características anfipáticas. O balanço entre grupos catiônicos e aniônicos da bactéria atribui um ponto isoelétrico (PIE) equivalente de 2,2. O perfil de potencial zeta da amostra mineral de hematita após interação com a bactéria mostrou uma mudança, onde o PIE mudou de 5,3 para 2,1. Para o estudo dos ensaios de adesão e microflotação, a amostra foi condicionada com a biomassa por meio de agitação sob condições específicas, tais como os tamanho de partícula, a concentração de biomassa, o pH da solução e tempo de condicionamento. A adesão da biomassa na superfície do mineral foi maior em pH 2 e na concentração de 200 mg / L. Os testes de microflotação foram feitos num tubo de Hallimond e foi avaliado a formação de espuma para uma concentração bacteriana de 200 mg / L, onde foi observado que a tensão superficial da solução aumenta à medida que o pH se torna básico. Das três faixas granulométricas utilizadas, a maior flotabilidade (83.86 por cento) foi alcançada na fração granulométrica (53 - 38 um), num pH 6 e com um tempo de flotação de 10 min. A bioflotação do mineral hematita segue o modelo cinético de segunda ordem, observou-se que as constantes de taxa (K2) da flotação do mineral aumentam com reduções de tamanho de partícula, mudando de 0,16369 (g.min)(-1) para 0,51604 (g.min)(-1) quando o tamanho de particula passou de (150 - 106 um) para (53 - 38 um). Os resultados apresentados mostram que o estudo do comportamento da cepa bacteriana Rhodococcus erythropolis como bioreagente na flotação de hematita foi viável, demonstrando o seu potencial uso como bioreagente coletor de mineral hematita, e assim projetando-se para uma futura aplicação na indústria da flotação mineral. / [en] The growing world demand for raw minerals has led to the increased mineral exploration and at the same time new research is being directed toward the production of new flotation reagents, so that they present higher selectivity and they are environmentally friendly. This research aimed to study the fundamental aspects of bioflotation of hematite, evaluating the bacterial strain Rhodococcus erythropolis as biocollector. Among the studies are conducted chemical analyzes to determine the proteins and carbohydrates present in the bacterial concentrate, it was established that is composed of macromolecules with amphipathic characteristics. The balance between cationic and anionic groups of the bacteria assigns an equivalent isoelectric point (IEP) of 2.2. The profile of zeta potential of the sample of hematite mineral after interaction with the bacteria showed a change, where the IEP has changed from 5.3 to 2.1. To study the adhesion and microflotation assays, the mineral sample was conditioned with the biomass by stirring under specific conditions, such as the particle sizes, biomass concentration, the pH of the solution and conditioning time. The biomass adhesion on mineral surface was higher at pH 2 and at the concentration of 200 mg / L. The microflotation tests were carried out in Hallimond tube and was evaluated the foam formation to a bacterial concentration of 200 mg / L, which was observed that the surface tension of the solution increases as the pH becomes basic. Of the three granulometric fractions used, the greatest floatability (83.86 percent) was achieved in the granulometric fraction (53 - 38 um), at pH 6 and with a flotation time of 10 min. The hematite mineral bioflotation follows the second-order kinetic model, was observed rate constant (K2) of the mineral flotation increase with reductions of particle size, moving from 0,16369 (g.min)(-1) for 0,51604 (g.min)(-1) when the particle size changed from (150 - 106 um) to (53 - 38 um). The results presented show that the study of the behavior of the bacterial 10 strain Rhodococcus erythropolis as bioreagent in the flotation of hematite was feasible, demonstrating its potential use as collector bioreagent of mineral hematite, and so projecting into a future application in the mineral flotation industry.

Im Rahmen der vorliegenden Arbeit wurden die Wechselwirkungen im System Bakterium –Tensid – Schadstoff mittels kalorimetrischer Untersuchungen (ITC, DSC) sowie mit XPS-Analysen und durch Zeta-Potential-Messungen an Bakterienoberflächen charakterisiert. Für die Untersuchungen wurden zwei Gram-positive Rhodococcus-Stämme und ein Gram-negativer Pseudomonas putida-Stamm verwendet. Als Biotenside wurden das Rhamnolipid JBR 425 und der von Rhodococcus erythropolis B7g produzierte Trehalosetetraester (THL-4) ausgewählt. Das synthetische Tensid SDS diente als Referenzsubstanz. Aus den kalorimetrischen Experimenten konnte eine starke Wechselwirkung zwischen den Tensiden und den aktiven Bakterienkulturen abgeleitet werden. THL-4 führte beim Wachstum der Rhodococcen auf n-Hexadecan zur Verkürzung der lag-Phase. SDS wies hingegen eine toxische Wirkung für die Bakterienstämme auf. Thermodynamische Betrachtungen ergaben, dass Wechselwirkungen des SDS mit den Bakterienzellen gegenüber der Mizellbildung bevorzugt werden.

Im Rahmen der vorliegenden Arbeit wurden die Wechselwirkungen im System Bakterium –Tensid – Schadstoff mittels kalorimetrischer Untersuchungen (ITC, DSC) sowie mit XPS-Analysen und durch Zeta-Potential-Messungen an Bakterienoberflächen charakterisiert. Für die Untersuchungen wurden zwei Gram-positive Rhodococcus-Stämme und ein Gram-negativer Pseudomonas putida-Stamm verwendet. Als Biotenside wurden das Rhamnolipid JBR 425 und der von Rhodococcus erythropolis B7g produzierte Trehalosetetraester (THL-4) ausgewählt. Das synthetische Tensid SDS diente als Referenzsubstanz. Aus den kalorimetrischen Experimenten konnte eine starke Wechselwirkung zwischen den Tensiden und den aktiven Bakterienkulturen abgeleitet werden. THL-4 führte beim Wachstum der Rhodococcen auf n-Hexadecan zur Verkürzung der lag-Phase. SDS wies hingegen eine toxische Wirkung für die Bakterienstämme auf. Thermodynamische Betrachtungen ergaben, dass Wechselwirkungen des SDS mit den Bakterienzellen gegenüber der Mizellbildung bevorzugt werden.

Biodesulfurization (BDS) is an alternative to hydrodesulfurization (HDS) as a method to remove sulfur from crude oil. Dibenzothiophene (DBT) was chosen as a model compound for the forms of thiophenic sulfur found in fossil fuels; up to 70% of the sulfur in petroleum is found as DBT and substituted DBTs; these compounds are however particularly recalcitrant to hydrodesulfurization, the current standard industrial method. My thesis deals with enhancing BDS through novel strains and through nanotechnology. Chapter highlights are: Chapter 2. My first aim was to isolate novel aerobic, mesophilic bacteria that can grow in mineral media at neutral pH value with DBT as the sole sulfur source. Different natural sites in Iran were sampled and I enriched, isolated and purified such bacteria. Twenty four isolates were obtained that could utilize sulfur compounds. Five of them were shown to convert DBT into HBP. After preliminary characterization, the five isolates were sent to the Durmishidze Institute of Biotechnology in Tbilisi for help with strain identification. Two isolates (F2 and F4) were identified as Pseudomonas strains, F1 was a Flavobacterium and F3 belonged to the strain of Rhodococcus. The definite identification of isolate F5 was not successful but with high probability it was a known strain. Since no new strains were apparently discovered, I did not work further in this direction. Chapter 3. In a second approach I studied the desulfurization ability of Shewanella putrefaciens strain NCIMB 8768, because in a previous investigation carried out at Cranfield University, it had been found that it reduced sulfur odour in clay. I compared its biodesulfurization activity profile with that of the widely studied Rhodococcus erythropolis strain IGTS8. However, S. putrefaciens was not as good as R. erythropolis. Chapter 4 and 5. I then turned to nanotechnology, which as a revolutionary new technological platform offers hope to solve many problems. There is currently a trend toward the increasing use of nanotechnology in industry because of its potentially revolutionary paths to innovation. I then asked how nanotechnology can contribute to enhancing the presently poor efficiency of biodesulfurization. Perhaps the most problematic difficulty is how to separate the microorganisms at the end of the desulfurization process. To make BDS more amenable, I explored the use of nanotechnology to magnetize biodesulfurizing bacteria. In other words, to render desulfurizing bacteria magnetic, I made them magnetic by decorating their outer surfaces with magnetic nanoparticles, allowing them to be separated using an external magnet. I used the best known desulfurizing bacterial strain, Rhodococcus erythropolis IGTS8. The decoration and magnetic separation worked very well. Unexpectedly, I found that the decorated cells had a 56% higher desulfurization activity compared to the nondecorated cells. I proposed that this is due to permeabilization of the bacterial membrane, facilitating the entry and exit of reactant and product respectively. Supporting evidence for enhanced permeabilization was obtained by Dr Pavel Grigoriev, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino. In Chapter 6, to optimize attachment of the nanoparticles to the surface of the bacteria I created thin magnetic nanofilms from the nanoparticles and measured the attachment of the bacteria using a uniquely powerful noninvasive optical technique (Optical Waveguide Lightmode Spectroscopy, OWLS) to quantify the attachment and determine how the liquid medium and other factors influence the process.