Spurred by a host of beneficial uses, the global use of nanoparticles is rapidly growing. Silver nanoparticles (Ag NPs) are used widely in consumer products, medicine, and the semiconductor industry. As nanoparticles become more commonly used, the transport of nanoparticles into the environment might negatively affect microorganisms in natural and engineered systems. The effects of Ag NPs on microorganisms have primarily been studied in planktonic or free-swimming cultures, but little work has been done to look at biofilm susceptibility to Ag NPs. This thesis describes bacterial tolerance, or the ability of an organism to survive exposure to an insult, to Ag NPs. The tolerance of planktonic and biofilm cells of the common wastewater treatment bacterium Zoogloea ramigera and a Rhodococcus strain isolated from drinking water was tested. These bacteria were exposed to different concentrations of Ag NPs, ranging from 0 to 25 mg/L, for a period of 5 hours. Results showed decreased tolerance with increasing Ag NP concentrations for both bacterial species. Z. ramigera biofilm cells are slightly more tolerant to Ag NPs than are planktonic cells. On the other hand, Rhodococcus planktonic and biofilm cells exhibit similar tolerance. However, in both cases, biofilm cells do not exhibit a striking protective effect against Ag NPs as compared to planktonic cells. This study shows that even short-term insults with Ag NPs can affect bacteria in engineered systems. A preliminary study of the shedding of free silver ions as a possible mechanism of Ag NP toxicity demonstrated that free silver ions were toxic to Escherichia coli in a 0.14M chloride environment. The data suggest that free silver ions can be pulled into solution from Ag NPs in chloride environments via ligand-promoted dissolution. Further work is needed to examine the antibacterial mechanism of Ag NPs against planktonic and biofilm cells to better understand how the release of nanoparticles into the environment can affect microorganisms in natural and engineered water systems. / text

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Bacteria have been confirmed as the main etiological factor for root canal infection as well as for root canal treatment failure. Thus the success of endodontic treatment depends on the complete elimination of bacteria and prevention of bacterial recolonization in the root canal system. The major challenge for conventional root canal disinfection strategies is the ability of bacteria to persist as biofilms within the anatomical complexities of the root canal system. In addition, the alterations in the ultrastructure of dentin tissue results in compromised structural integrity of root dentin leading to higher risk of fracture in root-filled teeth. The objectives of this study are twofold: 1) develop and test functionalized nanoparticles to eliminate biofilm bacteria and, 2) to stabilize and strengthen the dentin organic matrix by crosslinking collagen fibrils in the presence of biopolymeric nanoparticles. A bioactive polymeric nanoparticle functionalized with a photosensitizer may present as a single step treatment to achieve both the objectives. Chitosan a bioactive polymer was used owing to their inherent antibacterial and biocompatible characteristics. Chitosan micro-/nanoparticles were synthesized as well as functionalized with photosensitizer (rose bengal) for photodynamic activation. Bioactive chitosan nanoparticle functionalized with a rose bengal is expected to combine the properties of chitosan i.e., polycationic with higher affinity to bacterial cell wall and alter membrane integrity; that of a photosensitizer i.e., to generate singlet oxygen when photoactivated; and the nano-form further potentiate these specific properties. These photodynamically activable chitosan nanoparticles showed the distinct characteristics of chitosan and rose bengal. The synergistic effect of the chitosan conjugated nanoparticles was able to eliminate monospecies and multi-species bacterial biofilms with complete disruption of the biofilm structure. The singlet oxygen generated during photoactivation produced photochemical crosslinking of dentin collagen and infiltration of chitosan nanoparticles. Following crosslinking the dentin collagen showed significantly improved mechanical properties (ultimate tensile strength and toughness) and improved resistance to degradation by bacterial collagenase. In conclusion, this study presents a potential photosensitizer functionalized chitosan nanoparticles based treatment strategy to improve the success of endodontic treatment to achieve complete disinfection of the root canal system and enhanced the mechanical/ structural integrity of the root-filled teeth.

The human body is home for millions of bacteria. The largest microbial community is located in the gastro-intestinal (GI) tract, including the oral cavity with >700 identified taxa. Lactobacillus, which is normal inhabitant of the GI tract, contributes to health by possible biofilm and immune modulation. Breast milk is a claimed source for transmittance of Lactobacillus to infants’ GI tract, but there is limited information if breastfeeding affects lactobacilli in the oral cavity. The objectives of Papers I and II of this dissertation were to compare infant oral microbiota by feeding mode, and to characterize oral lactobacilli including potential probiotic properties of the dominant Lactobacillus species. Two cohorts with a total of 340 healthy 3- to 4-month-old infants were investigated. Saliva and oral mucosal swab samples were collected. Bacteria were characterized by culture-dependent and -independent methods, including 16S rRNA genes sequencing, quantitative PCR, and the Human Microbe Identification Microarray (HOMIM). Inhibition of growth and adhesion were also tested. Multivariate modeling of HOMIM-detected oral bacteria clustered breastfed infants separately from formula-fed infants, and linked breastfed infants to a more health-associated flora. Lactobacilli were essentially detected in breastfed infants only. Lactobacillus gasseri was most prevalent out of six identified Lactobacillus species. Infant isolates of L. gasseri bound to saliva gp340 and MUC7 and adhered to gingival epithelial cells. Infant isolates also inhibited adhesion of Streptococcus mutans to saliva-coated hydroxyapatite, and inhibited growth of S. mutans, Streptococcus sobrinus, Actinomyces naeslundii, Actinomyces oris, Candida albicans and Fusobacterium nucleatum in a concentration-dependent fashion. Papers III and IV aimed to assess persistence of probiotic Lactobacillus reuteri, if persistence is necessary for a regrowth of mutans streptococci (MS), and if L. reuteri intake affects oral microbiota composition. Two well-documented L. reuteri strains (DSM 17938 and PTA 5289) were used in two double-blind, randomized controlled trials. In the first, 62 subjects (test=32, placebo=30) with high counts of MS were exposed to L. reuteri for 6 weeks. Exposure followed full-mouth disinfection with chlorhexidine. In the second study, 44 healthy subjects (test=22, placebo=22) consumed the L. reuteri for 12 weeks. Saliva and biofilm samples were collected before, during and up to 6 months after exposure. Analyses included culture, strain-specific PCR and 454-pyrosequencing targeting the hypervariable region V3-V4 of the 16S rRNA gene. L. reuteri test strains were detected in the mouth of approximately two thirds of test participants during intake. However, their presence decreased gradually when consumption stopped. Subjects with detectable L. reuteri had slower regrowth of MS compared to non-carriers. Pyrosequencing yielded a total of 812,547 high-quality sequencing reads. Firmicutes, Proteobacteria, Bacteroidetes, Fusobacteria and Actinobacteria were the major bacterial phyla recovered. Exposure to L. reuteri strains did not affect overall phylotype abundance, but multivariate modeling clustered 12-week-treated test subjects separately from those who received placebo. Exposure to the test strains was strongly associated with presence and increased levels of F. nucleatum and Streptococcus spp. In conclusion, the oral microbiota differed by feeding mode in infants. One third of breastfed infants had lactobacilli in the mouth, while only single formula-fed infant had it. L. gasseri, predominant in infants, displayed probiotic characteristics in vitro. Retention of probiotic L. reuteri was a prerequisite for delay of MS regrowth after disinfection. However, probiotic bacteria may not be beneficial for all, since L. reuteri DSM 17938 and PTA 5289 were retained in only 2 of 3 consumers. Finally, the altered microbiota after 12 weeks consumption of L. reuteri indicates that intake of probiotic bacteria, or at least L reuteri, has an impact on oral ecology. However, this finding needs further investigation. / Vår kropp består av fler mikroorganismer än egna celler.  De miljontals bakterier som finns på ut - och insidan av kroppen är som regel harmlösa och vissa är till och med till nytta för oss. Magtarmkanalen, som startar med munnen, är den kroppsdel som härbärgerar flest bakterier. Till exempel har man bara i munnen identifierat totalt mer än 700 olika arter. En av dessa är Lactobacillus, en bakterieart som finns i normalfloran och som har probiotiska egenskaper. Hos spädbarn anses bröstmjölk vara en källa för Lactobacillus i tarmen, men hur amning påverkar laktobacillförekomst i munnen är oklart. Den första delen i denna avhandling syftar till att jämföra mikrofloran i munnen hos spädbarn som ammas kontra de som får ersättning, att karakterisera vilka laktobaciller som finns i munnen hos respektive grupp och undersöka om dessa har probiotiska egenskaper. Totalt studerades saliv och prov från munslemhinnan från 340 friska 3-4 månader gamla spädbarn. Proven karakteriserades med odling, sekvensering, kvantitativ PCR och en microarraymetod (Human Microbe Identification Microarray, HOMIM), och isolerade laktobacillers effekt på växt och vidhäftning av andra munbakterier studerades. Ammade barn hade en mer hälsoassocierad mikroflora i munnen. Laktobaciller fanns bara hos ammade barn, men bara hos vart 3:e ammat barn. Av totalt sex identifierade laktobacillarter var Lactobacillus gasseri den i särklass mest förekommande arten. L. gasseri isolerade från spädbarnen band till salivproteinerna gp-340 och MUC7 samt till orala epitelceller. L. gasseri kunde även förhindra adhesion av Streptococcus mutans till konstgjord tandemalj och hämma växt av S. mutans, Streptococcus sobrinus, Actinomyces naeslundii, Actinomyces oris, Candida albicans och Fusobacterium nucleatum. Laktobaciller förekommer i många hälsoprodukter med påstådd probiotisk effekt. Andra delen av denna avhandling syftade till att bedöma om intag av tabletter med den probiotiska arten Lactobacillus reuteri påverkar ekologin i mikrofloran i munnen, om arten etablerar sig hos alla vid exponering, och om etablering är nödvändig för probiotisk effekt (mätt som hämmad återväxt av kariesassocierade mutansstreptokocker efter antimikrobiell behandling). Två stammar L. reuteri (DSM 17938 and PTA 5289) användes i två dubbelblinda, randomiserade studier. I båda studierna intog deltagarna i testgruppen tabletter med L. reuteri-stammarna och de i kontrollgruppen identiska tabletter utan bakterier. I den första studien deltog 62 deltagare (32 test, 30 kontroll) i 6 veckor och i den andra 44 personer (22 test, 22 placebo) under 12 veckor. Saliv och biofilmsprover samlades in vid studiestart, under och upp till 6 månader efter avslutad testperiod. Proverna analyserades med odling, PCR och 454-pyrosekvensering. L. reuteri etablerade sig hos 2/3 av testpersonerna under testperioden men mängden minskade gradvis efter avslutat intag. Bland de som fick L. reuteri hade deltagarna med påvisbara teststammar fördröjd återväxt av mutansstreptokocker jämfört med de som inte hade det. Pyrosekvensering visade att totalantalet phylotyper inte skiljde sig mellan de som fick aktiva kontra placebotabletter, men att ekologin i bakteriefilmerna hos de som ätit de aktiva tabletterna ändrades. Att exponeras för L. reuteri var starkt associerat med förhöjda nivåer av F. nucleatum and Streptococcus spp. Sammanfattningsvis visar dessa studier att amning är associerad med att ha probiotiska laktobaciller i munnen men bara vissa etablerar arten i munnen. Hos vuxna försenade L. reuteri återkolonisation av mutansstreptokocker efter antibakteriell behandling, och påverkade ekologin i bakteriefilmerna i munnen. Även hos vuxna ledde exponering till etablering bara hos vissa individer.

Listeria monocytogenes has been found to withstand harsh environmental conditions including desiccation. The pathogen is also known to form biofilm when in co-culture with other bacteria found in food products. This study investigated the desiccation survival of L. monocytogenes in mixed biofilms with Pseudomonas fluorescens, Serratia liquefaciens and Shewanella putrefaciens. To this end, mono- or binary species biofilms were formed and desiccated (43% relative humidity, 21 days at 15°C) on stainless steel coupons and the double Weibull model was fitted to the resulting survivor curves. The presence of the competitor Gram-negative food spoilage bacteria with the exception of Sh. putrefaciens suppressed (p<0.05) L. monocytogenes during biofilm formation (100% relative humidity, 15°C and 48 h) and subsequently decreased (P<0.05) the desiccation survival in L. monocytogenes without affecting the resistance of individual cells. Microscopic approaches revealed different biofilm forming capabilities in the mono- and binary bacterial combinations.

The goal of this study was to investigate the effects of intertidal mudflat biofilms on sediment erosion in the Minas Basin of the Bay of Fundy, Canada. From April through November 2012, sediment cores were collected biweekly and eroded using a Gust micro- cosm. Half of the cores were eroded without undergoing prior treatment, while the other half were treated with bleach prior to erosion to destroy biofilms. Size-specific sediment retention by biofilms was evaluated by comparing the disaggregated inorganic grain size (DIGS) distributions of sediment resuspended from untreated and treated cores, while seasonal variation in natural sediment erodibility was assessed by focusing on the mass eroded from untreated cores only. Results show that biofilms preferentially retained clays and very fine silts (< 10 μm), and that overall sediment erodibility decreased from spring to fall. Results also indicate that abundance of the infaunal amphipod Corophium volutator and rainfall increased sediment erodibility.

Bacteria have been confirmed as the main etiological factor for root canal infection as well as for root canal treatment failure. Thus the success of endodontic treatment depends on the complete elimination of bacteria and prevention of bacterial recolonization in the root canal system. The major challenge for conventional root canal disinfection strategies is the ability of bacteria to persist as biofilms within the anatomical complexities of the root canal system. In addition, the alterations in the ultrastructure of dentin tissue results in compromised structural integrity of root dentin leading to higher risk of fracture in root-filled teeth. The objectives of this study are twofold: 1) develop and test functionalized nanoparticles to eliminate biofilm bacteria and, 2) to stabilize and strengthen the dentin organic matrix by crosslinking collagen fibrils in the presence of biopolymeric nanoparticles. A bioactive polymeric nanoparticle functionalized with a photosensitizer may present as a single step treatment to achieve both the objectives. Chitosan a bioactive polymer was used owing to their inherent antibacterial and biocompatible characteristics. Chitosan micro-/nanoparticles were synthesized as well as functionalized with photosensitizer (rose bengal) for photodynamic activation. Bioactive chitosan nanoparticle functionalized with a rose bengal is expected to combine the properties of chitosan i.e., polycationic with higher affinity to bacterial cell wall and alter membrane integrity; that of a photosensitizer i.e., to generate singlet oxygen when photoactivated; and the nano-form further potentiate these specific properties. These photodynamically activable chitosan nanoparticles showed the distinct characteristics of chitosan and rose bengal. The synergistic effect of the chitosan conjugated nanoparticles was able to eliminate monospecies and multi-species bacterial biofilms with complete disruption of the biofilm structure. The singlet oxygen generated during photoactivation produced photochemical crosslinking of dentin collagen and infiltration of chitosan nanoparticles. Following crosslinking the dentin collagen showed significantly improved mechanical properties (ultimate tensile strength and toughness) and improved resistance to degradation by bacterial collagenase. In conclusion, this study presents a potential photosensitizer functionalized chitosan nanoparticles based treatment strategy to improve the success of endodontic treatment to achieve complete disinfection of the root canal system and enhanced the mechanical/ structural integrity of the root-filled teeth.

Los ríos están amenazados por presiones socioeconómicas que alteran los usos del suelo y el clima degradando las condiciones ambientales, por ende afectando la calidad del agua y la hidrología de estos ecosistemas. Aproximadamente el 40% de la superficie terrestre está ocupada por cultivos y pasturas, y el uso de fertilizantes que contienen fósforo y nitrógeno se ha incrementado entre dos y siete veces respectivamente en los últimos 40 años. Estos cambios forman parte de lo que se conoce como “cambios globales”, cuya definición abarca la combinación de cambios climáticos (tales como alteración de los patrones de precipitación y temperatura, variaciones en el CO<SUB>2</SUB> atmosférico, etc.), y de cambios en el uso de la tierra (como el aumento en el uso de fertilizantes, aumento de la urbanización, etc.). Por lo tanto es esperable de acuerdo con los futuros escenarios de cambios climáticos que los cursos de agua sufran cambios de distinta magnitud, dependiendo el área del planeta que se analice. En el caso de la región pampeana estos incluirían de acuerdo con los pronósticos aumentos en la escorrentía y temperatura que acarrearán aumentos de nutrientes, sólidos en suspensión y de la velocidad de la corriente. De acuerdo con los descriptores analizados en las experiencias in situ y ex situ realizadas en esta tesis, fue posible comprobar algunos de los efectos pertinentes a estas problemáticas en sistemas lóticos pampeanos. En esta tesis se focalizó el interés en el desarrollo y las características del biofilm epipélico, que recubre los sedimentos finos del lecho de los cursos de agua pampeanos. Esta microcomunidad está modulada por muchos factores, entre los que se incluyen las concentraciones de nutrientes inorgánicos, la luz, temperatura, el sustrato, la herbivoría, la velocidad de la corriente y las características de las partículas transportadas por el flujo, entre otros. Por lo tanto, las presiones originadas por los cambios globales pueden tener efectos significativos sobre el desarrollo de este tipo de biofilm. El objetivo general de la presente tesis fue estudiar los cambios en la estructura y funcionamiento de los biofilms epipélicos de cursos de agua de la llanura pampeana expuestos a las consecuencias relacionadas con algunos de los efectos de los cambios globales. Para esto se realizaron experiencias in situ y ex situ con la finalidad de contrastar hipótesis vinculadas al incremento de nutrientes, temperatura, turbidez y velocidad de la corriente, como algunas de las variables que podrán ser afectadas en el marco de estos cambios. Para cumplir esta finalidad se realizó una experiencia de adición de nutrientes en campo y dos experiencias de laboratorio en canales artificiales. La experiencia de adición experimental de nutrientes (fósforo y nitrógeno) se realizó en un tramo de un arroyo pampeano (denominado tramo Impacto), y se comparó con un tramo del mismo arroyo sin modificar (denominado tramo Control). Ambos tramos fueron muestreados por 9 meses previamente a la adición experimental de nutrientes y durante 12 meses durante la adición continua de nutrientes. En esta experiencia, llevada a cabo en el Arroyo “La Choza” de la localidad de Luján, se estudiaron las variaciones en las biomasas algales y bacterianas y la producción de enzimas relacionadas con la degradación de materia orgánica (&beta;-glucosidasa) y con la adquisición de nutrientes (fosfatasa). Para esta fertilización se buscó generar un enriquecimiento moderado, triplicando los niveles basales del arroyo de fósforo y nitrógeno, para lo que se adicionó fertilizante de uso comercial (Nitrofoska Azul ® 12%P, 12%N). Entre los resultados obtenidos, se observó un aumento progresivo de la biomasa algal seguido por aumentos graduales de la biomasa bacteriana. Esto sugiere que las bacterias basan su consumo preferentemente en los productos autotróficos (ej.: carbono) mientras que el consumo de nutrientes inorgánicos tanto como para algas como para bacterias es reducido. Por otro lado, la falta de respuesta de las enzimas analizadas a la adición de nutrientes, y su baja concentración en el arroyo, demuestran que el biofilm no es estimulado por estas condiciones para producirlas. Esto es consistente con los arroyos donde los nutrientes no son limitantes, y su adquisición resulta simplificada. Las dos experiencias en laboratorio se llevaron a cabo empleando canales artificiales. Una se realizó con un biofilm proveniente de un arroyo con bajo impacto antropogénico (valores promedio de Fósforo Reactivo Soluble (PRD) 0,37 mgL-1; Nitrógeno Inorgánico Disuelto (NID) 0,22 mgL-1, DBO5 6 mgL-1, DQO 15 mgL-1, OD 7,70 mgL-1) y otra con uno procedente de un arroyo con alto impacto antropogénico (valores promedio de PRS 2,24 mgL-1; NID 1,85 mgL-1, DBO5 46 mgL-1, DQO 105 mgL-1, OD 5,28 mgL-1). El biofilm de ambos arroyos se desarrolló en los canales y se les aplicó dos tipos de tratamientos (TB: tratamiento bajo y TA: tratamiento alto). Estos consistieron en incrementar los nutrientes en el agua (fósforo y nitrógeno), la turbidez, la velocidad de la corriente y la temperatura de acuerdo con las predicciones planteadas en los escenarios de cambio global. El tratamiento TA recibió un mayor incremento de esas variables que el tratamiento TB, y ambos tratamientos fueron comparados con canales control (C). Cada tratamiento y control fue realizado por triplicado, y el agua de los canales artificiales fue provista por el mismo arroyo de donde se colectaron los biofilms. Las experiencias de laboratorio presentadas en esta tesis trataron de demostrar que el efecto combinado del aumento de la velocidad de la corriente, nutrientes, turbidez y temperatura, favorecen un mayor desarrollo y actividad en la comunidad biológica. Los principales resultados evidencian que la composición de especies del biofilm es alterada cuando es expuesta a los efectos simultáneos de estos cuatro estresores asociados a los cambios globales en la llanura pampeana. La disminución de especies de diatomeas oligotróficas y oligosaprobicas en el experimento con biofilms del arroyo con bajo impacto antropogénico indica que el ensamble es afectado por los tratamientos. En esa misma experiencia, la diversidad y la riqueza específica disminuyeron en los tratamientos más intensos del experimento, sugiriendo que dicha comunidad es más sensible que la desarrollada en el experimento con biofilms del arroyo mas impactado. Sin embargo, otras variables estructurales y metabólicas que caracterizan el biofilm en general – tales como el contenido de clorofila “a”, la biomasa bacteriana, los carbohidratos totales y el peso seco libre de cenizas– no evidenciaron diferencias significativas a causa de los estresores estudiados. Hay que considerar que las pequeñas variaciones debidas a los tratamientos pueden ser importantes para la escala de tiempo empleada, e incluso la estructura de las comunidades fue alterada hasta la significancia estadística. Por ejemplo, las proporciones de especies de diatomeas, al ser clasificadas por su consumo del nitrógeno, saprobiedad y estado trófico, sufrieron pequeñas modificaciones en el corto plazo de las experiencias. Estos cambios estructurales en estas comunidades fueron mayores en aquella que provenía de un arroyo con un menor impacto antropogénico, como es esperable a comunidades menos tolerantes. Estos cambios parecen sutiles, pero resultan amplios si se considera la escala temporal y espacial utilizada. Los cambios globales ejercen sus efectos a escalas temporales y espaciales mucho mayores a las utilizadas en estos ensayos, y la cantidad de variables a considerar para su estudio apropiado es muy vasta. Pero es mediante el estudio a escalas pequeñas que se puede llegar a una mejor comprensión de la dinámica interna de esta microcomunidad y de los efectos que tienen las variables que influencian su desarrollo. De acuerdo con los resultados obtenidos en esta tesis es posible reconocer que los arroyos pampeanos demuestran una resistencia al cambio que se manifiesta a través de respuestas diferidas o de pequeños cambios visibles a diferentes escalas. En síntesis, la mayor parte de las hipótesis planteadas en esta tesis fueron rechazadas. Por lo cual el efecto de los enriquecimientos con nutrientes puede ser menos predecible en sistemas con altas concentraciones basales de nutrientes, como es el caso de los arroyos pampeanos, naturalmente enriquecidos. Referencias bibliográficas acreditan que estos arroyos muestran una red trófica muy compleja y compartimentalizada, y esa complejidad sería una de las causas que amortiguarían la transferencia potencial de los efectos de las adiciones de nutrientes. Estos resultados indican que el amplio rango de medidas que pueden reducir los efectos de la eutrofización en arroyos debería incluir la planificación de los usos del suelo, particularmente los usos urbanos y agrícolas, reconocidos como las principales fuentes de nutrientes que pueden ingresar a los arroyos.

In this work, a differential reactor was used to expose all the biofilter packing material (compost) to a uniform toluene concentration in air. The reactor was combined with water content control using the suction cell principle and traditional inlet concentration, temperature and humidity control. The matric potential was controlled using the suction cell principle between -5 to -300 cm H₂O which controlled the water content between 0.99 and 2.30 g g⁻¹ (dry weight). Two types of compost were used, with different water retention curves with no observed difference in elimination capacity. The elimination capacity varied between 2.7 g m⁻³r hr⁻¹ and 21 g m⁻³r hr⁻¹ with low potential causing low removal rates. The reduction in EC at low matric potentials was attributed to several factors: loss of water availability to the organisms, water redistribution in the medium, non-adaptable micro-organisms, and reduced mass transfer. Cultures isolated from compost were used to inoculate the reactor to create a biofilm. A maximal observed surface EC of is 0.17 g m⁻²r hr⁻¹ and a specific removal rate of 1250 g m⁻³b hr⁻¹ is measured. These values were used in modelling the biofilter performance. The EC was dependent on the residual toluene concentration. The EC increased with increasing toluene concentration until reaching a critical concentration. Above this concentration, 100 – 300 ppm (0.37- 1.11 g m⁻³) depending on biofilm thickness and area of coverage, the EC was constant. Three toluene dependency curves were fitted using a zero order and a composite model using a weighted average of a zero and first order component. From the data the critical concentration (Ccrit) and the ECcrit was found and used to determine the biofilm thickness. It was estimated to be between 68 and 134 µm. Using a qmax of 1250 g m⁻³b hr⁻¹ and optimising the model a Ks of 1.3•10⁻¹ g m⁻³g was found. This was comparable to values found in the literature. There was no significant difference in the fit between both models. The Ks was low compared to the majority of the data, which means that the zero order part of the composite model dominated. Nitrogen and other nutrients were added to investigate their influence on the elimination capacity (EC) of toluene. Also the effect of temperature on the EC was investigated between 14 and 60 °C. Maximal removal rates were found between 25 and 55 °C. The EC decreased by 90% going from 55 to 60 °C and took many weeks to recover. Without any extra nitrogen added to the media, the EC averaged around 6 ± 0.3 g m⁻³r h⁻¹. Although the average EC was lower than most reports for toluene removal, it was still in the general range reported. When NH4Cl (1 g l⁻¹) was added to the reactor, the EC increased to 41 ± 1.7 g m⁻³r hr⁻¹. Similar effects were observed with nitrate addition; the steady state EC doubled from 30.1 ± 0.9 g m⁻³r hr⁻¹ to 76.3 ± 2.5 g m⁻³r hr⁻¹. Other macronutrients tested like phosphate, sulphate, magnesium, calcium and iron did not increase the EC.