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Controlling the morphology of filamentous fungi is crucial to improve the performance of fungal bioprocesses. Microparticle-enhanced cultivation (MPEC) increases productivity, most likely by changing the fungal morphology. However, due to a lack of appropriate methods, the exact impact of the added microparticles on the structural development of fungal pellets is mostly unexplored. In this study synchrotron radiation-based microcomputed tomography and three-dimensional (3D) image analysis were applied to unveil the detailed 3D incorporation of glass microparticles in nondestructed pellets of Aspergillus niger from MPEC. The developed method enabled the 3D analysis based on 375 pellets from various MPEC experiments. The total and locally resolved volume fractions of glass microparticles and hyphae were quantified for the first time. At increasing microparticle concentrations in the culture medium, pellets with lower hyphal fraction were obtained. However, the total volume of incorporated glass microparticles within the pellets did not necessarily increase. Furthermore, larger microparticles were less effective than smaller ones in reducing pellet density. However, the total volume of incorporated glass was larger for large microparticles. In addition, analysis of MPEC pellets from different times of cultivation indicated that spore agglomeration is decisive for the development of MPEC pellets. The developed 3D morphometric analysis method and the presented results will promote the general understanding and further development of MPEC for industrial application.
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The actinomycete Lentzea aerocolonigenes produces the antitumor antibiotic rebeccamycin. In previous studies the rebeccamycin production was significantly increased by the addition of glass beads during cultivation in different diameters between 0.5 and 2 mm and the induced mechanical stress by the glass beads was proposed to be responsible for the increased production. Thus, this study was conducted to be a systematic investigation of different parameters for macroparticle addition, such as bead diameter, concentration, and density (glass and ceramic) as well as shaking frequency, for a better understanding of the particle-induced stress on L. aerocolonigenes. The induced stress for optimal rebeccamycin production can be estimated by a combination of stress energy and stress frequency. In addition, the macroparticle-enhanced cultivation of L. aerocolonigenes was combined with soy lecithin addition to further increase the rebeccamycin concentration. With 100 g L-1 glass beads in a diameter of 969 µm and 5 g L-1 soy lecithin a concentration of 388 mg L-1 rebeccamycin was reached after 10 days of cultivation, which corresponds to the highest rebeccamycin concentrations achieved in shake flask cultivations of L. aerocolonigenes stated in literature so far.
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Actinobacteria/crescimento & desenvolvimento , Carbazóis/metabolismo , Vidro , Lecitinas/farmacologia , Estresse Mecânico , Lecitinas/metabolismoRESUMO
A new disposable, multiphase, microbioreactor (MBR; with a working volume of 550 µl) equipped with online sensors is presented for biotechnological screening research purposes owing to its high-throughput potential. Its design and fabrication, online sensor integration, and operation are described. During aerobic cultivation, sufficient oxygen supply is the most important factor that influences growth and product formation. The MBR is a microbubble column bioreactor (µBC), and the oxygen supply was realized by active pneumatic bubble aeration, ensuring sufficient volumetric liquid-phase mass transfer (k L a) and proper homogenization of the cultivation broth. The µBC was equipped with miniaturized sensors for the pH, dissolved oxygen, optical density and glucose concentration that allowed real-time online monitoring of these process variables during cultivation. The challenge addressed here was the integration of sensors in the limited available space. The MBR was shown to be a suitable screening platform for the cultivation of biological systems. Batch cultivations of Saccharomyces cerevisiae were performed to observe the variation in the process variables over time and to show the robustness and operability of all the online sensors in the MBR.
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Produtos Biológicos/metabolismo , Reatores Biológicos/microbiologia , Biotecnologia/métodos , Programas de Rastreamento/métodos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Aerobiose , Meios de Cultura/química , Glucose/análise , Concentração de Íons de Hidrogênio , Oxigênio/análise , EspectrofotometriaRESUMO
Rebeccamycin is an antibiotic and antitumor substance isolated from the filamentous bacterium Lentzea aerocolonigenes. After its discovery, investigations of rebeccamycin focused on elucidating its structure, biological activity, and biosynthetic pathway. For potential medical application, a sufficient drug supply has to be ensured, meaning that the production process of rebeccamycin plays a major role. In addition to the natural production of rebeccamycin in L. aerocolonigenes, where the complex cell morphology is an important factor for a sufficient production, rebeccamycin can also be heterologously produced or chemically synthesized. Each of these production processes has its own challenges, and first approaches to production often lead to low final product concentrations, which is why process optimizations are performed. This review provides an overview of the production of rebeccamycin and the different approaches used for rebeccamycin formation including process optimizations.
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Antibióticos Antineoplásicos/biossíntese , Bactérias/metabolismo , Carbazóis/metabolismo , Microbiologia Industrial , Antibióticos Antineoplásicos/síntese química , Antibióticos Antineoplásicos/química , Bactérias/genética , Carbazóis/síntese química , Carbazóis/químicaRESUMO
BACKGROUND: Different strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv. RESULTS: Extracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L-1) and highest space-time-yield (8 mg L-1 h-1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac). CONCLUSIONS: The genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space-time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains.
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Bacillus megaterium/imunologia , Bacillus/imunologia , Proteínas Recombinantes/biossíntese , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Bacillus/classificação , Bacillus/genética , Bacillus/metabolismo , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas de Bactérias/genética , Reatores Biológicos , Meios de Cultura , Microbiologia Industrial/métodos , Penicilina Amidase/genética , Penicilina Amidase/metabolismo , Peptídeo Hidrolases/metabolismo , Plasmídeos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/análise , Anticorpos de Cadeia Única/imunologiaRESUMO
Over the past decades a large amount of biopolymers originating from various types of microorganisms have been reported. With ongoing research the number of possible applications has increased rapidly, ranging from use as food additives and biomedical agents to biodegradable plastics from renewable resources. In spite of the plethora of applications, the large-scale introduction of biopolymers into the market has often been forestalled by high production costs mainly due to complex or inefficient downstream processing. In this article, state-of-the-art methods and recent advances in the separation and purification of microbial polymers are reviewed, with special focus on the biopolymers, γ-polyglutamic acid and xanthan gum. Furthermore, a study of the general factors affecting production and purification is presented, including biopolymer rheology, enzymatic degradation and production of biopolymer mixtures.
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Biopolímeros , Biotecnologia , Microbiologia Industrial , Ácido Poliglutâmico , Polissacarídeos BacterianosRESUMO
This paper presents the applicability of a microtechnologically fabricated microbubble column as a screening tool for submerged aerobic cultivation. Bubbles in the range of a few hundred micrometers in diameter were generated at the bottom of an upright-positioned microdevice. The rising bubbles induced the circulation of the liquid and thus enhanced mixing by reducing the diffusion distances and preventing cells from sedimentation. Two differently sized nozzles (21 × 40 µm(2) and 53 × 40 µm(2) in cross-section) were tested. The gas flow rates were adjustable, and the resulting bubble sizes and gas holdups were investigated by image analysis. The microdevice features sensor elements for the real-time online monitoring of optical density and dissolved oxygen. The active aeration of the microdevice allowed for a flexible oxygen supply with mass transfer rates of up to 0.14 s(-1). Slightly higher oxygen mass transfer rates and a better degassing were found for the microbubble column equipped with the smaller nozzle. To validate the applicability of the microbubble column for aerobic submerged cultivation processes, batch cultivations of the model organism Saccharomyces cerevisiae were performed, and the specific growth rate, oxygen uptake rate, and yield coefficient were investigated.
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Reatores Biológicos/microbiologia , Oxigênio/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , AerobioseRESUMO
For the commercially established process of paclitaxel production with Taxus chinensis plant cell culture, the size of plant cell aggregates and phenotypic changes in coloration during cultivation have long been acknowledged as intangible parameters. So far, the variability of aggregates and coloration of cells are challenging parameters for any viability assay. The aim of this study was to investigate simple and non-toxic methods for viability determination of Taxus cultures in order to provide a practicable, rapid, robust and reproducible way to sample large amounts of material. A further goal was to examine whether Taxus aggregate cell coloration is related to general cell viability and might be exploited by microscopy and image analysis to gain easy access to general cell viability. The Alamar Blue assay was found to be exceptionally eligible for viability estimation. Moreover, aggregate coloration, as a morphologic attribute, was quantified by image analysis and found to be a good and traceable indicator of T. chinensis viability.
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Colorimetria , Taxus/citologia , Reprodutibilidade dos Testes , Estresse MecânicoRESUMO
Due to the overuse of antibiotics, the number of multidrug-resistant pathogen bacteria is rising in recent years posing a serious threat to human health. One promising alternative for treatment is the application of phage therapy using highly selective bacteriophages. Because of their selectivity, individual screens called phagograms for each patient are required to select phages from a phage library. Phagograms are mostly performed via bacterial cultivation on double layer agar plates and phage addition causing bacterial lysis. However, these assays are work-intensive and have a low ability for parallelization and automation. Hence, highly parallelizable and automatable microbioreactors in the lowest microliter scale could offer an economic solution increasing the throughput of phagograms. This paper demonstrates the applicability of a novel capillary-wave microbioreactor (cwMBR) to perform phagograms. Due to its small volume of only 7µL and the open-droplet design, it can be easily automated and parallelized in future. Furthermore, the ability of online biomass measurement makes the cwMBR a perfect phagogram platform in the future. Herein, phagograms with E. coli and different concentrations of the phages MM02 and EASG3 were performed as proof of concept for phagograms in the cwMBR. Thereby, the cwMBR was able to measure differences in lysis kinetics of different phages. Furthermore, the phagograms were compared to those in conventional microtiter plate readers revealing the cwMBR as ideal alternative for phagograms as it combines favorable mixing conditions and a phage repellent hydrophilic glass surface with online biomass measurement in an open-droplet design for future parallelization and automation.
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In recent years, the advances in microbiology show that biofilms are structurally complex, dynamic and adaptable systems including attributes of multicellular organisms and miscellaneous ecosystems. One may distinguish between beneficial and harmful biofilms appearing in daily life as well as various industrial processes. In order to advance the growth of the former or prevent the latter type of biofilm, a detailed understanding of its properties is indispensable. Besides microbiological aspects, this concerns the determination of mechanical characteristics, which provides the basis for material modelling. In the present paper the existing experimental methods that have been proposed since the 1980s are reviewed and critically discussed with respect to their usefulness and applicability to develop numerical modelling approaches.
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Biofilmes , Modelos Biológicos , Fenômenos BiomecânicosRESUMO
In response to sudden decrease in osmotic pressure, halophilic microorganisms secrete their accumulated osmolytes. This specific stress response, combined with physiochemical responses to the altered environment, influence the membrane properties and integrity of cells, with consequent effects on growth and yields in bioprocesses, such as bacterial milking. The aim of this study was to investigate changes in membrane fluidity and integrity induced by environmental stress in ectoine-secreting organisms. The halophilic ectoine-producing strains Alkalibacillus haloalkaliphilus and Chromohalobacter salexigens were treated hypo- and hyper-osmotically at several temperatures. The steady-state anisotropy of fluorescently labeled cells was measured, and membrane integrity assessed by flow cytometry and ectoine distribution. Strong osmotic downshocks slightly increased the fluidity of the bacterial membranes. As the temperature increased, the increasing membrane fluidity encouraged more ectoine release under the same osmotic shock conditions. On the other hand, combined shock treatments increased the number of disintegrated cells. From the ectoine release and membrane integrity measurements under coupled thermal and osmotic shock conditions, we could optimize the secretion conditions for both bacteria.
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Diamino Aminoácidos/metabolismo , Chromohalobacter/metabolismo , Fluidez de Membrana , Chromohalobacter/crescimento & desenvolvimento , Corantes Fluorescentes , Temperatura Alta , OsmoseRESUMO
In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular morphology are often closely linked, requiring a well-defined level of mechanical stress to achieve high product concentrations. Macroparticles were added to shake flask cultures of the filamentous actinomycete Lentzea aerocolonigenes to find these optimal cultivation conditions. However, there is currently no model concept for the dependence of the strength and frequency of the bead-induced stress on the process parameters. Therefore, shake flask simulations were performed for combinations of bead size, bead concentration, bead density and shaking frequency. Contact analysis showed that the highest shear stresses were caused by bead-bottom contacts. Based on this, a newly generated characteristic parameter, the stress area ratio (SAR), was defined, which relates the bead wall shear and normal stresses to the total shear area. Comparison of the SAR with previous cultivation results revealed an optimum pattern for product concentration and mean product-to-biomass related yield coefficient. Thus, this model is a suitable tool for future optimization, comparison and scaling up of shear-sensitive microorganism cultivation. Finally, the simulation results were validated using high-speed recordings of the bead motion on the bottom of the shake flask.
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Lentzea aerocolonigenes, as an actinomycete, is a natural producer of the antibiotic and antitumoral drug rebeccamycin. Due to the filamentous cellular morphology handling in cultivations is challenging; therefore, morphology engineering techniques are mandatory to enhance productivity. One promising approach described in the literature is the addition of mineral particles in the micrometer range to precisely adjust cellular morphology and the corresponding product synthesis (microparticle-enhanced cultivation, MPEC). Glass microparticles are introduced in this study as a novel supplementation type for bioprocess intensification in filamentous organisms. Several investigations were conducted to screen for an optimal particle setup, including particle size and concentration regarding their impact and effects on enhanced productivity, microparticle incorporation behavior into the biopellets, the viability of pellets, and morphological changes. Glass microparticles (10 g·L-1) with a median diameter of 7.9 µm, for instance, induced an up to fourfold increase in product synthesis accompanied by overall enhanced viability of biomass. Furthermore, structural elucidations showed that biopellets isolated from MPEC tend to have lower hyphal density than unsupplemented control pellets. In this context, oxygen microprofiling was conducted to better understand how internal structural changes interwind with oxygen supply into the pellets. Here, the resulting oxygen profiles are of a contradictive trend of steeper oxygen consumption with increasing glass microparticle supplementation. Eventually, MPEC was combined with another promising cultivation strategy, the supplementation of soy lecithin (7.5 g·L-1), to further increase the cultivation performance. A combination of both techniques in an optimized setup resulted in a rebeccamycin concentration of 213 mg·L-1 after 10 days of cultivation, the highest value published so far for microparticle-supplemented shake flask cultivations of L. aerocolonigenes.
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Functional nanofibrils from globular proteins are usually formed by heating for several hours at pH 2.0, which induces acidic hydrolysis and consecutive self-association. The functional properties of these micro-metre-long anisotropic structures are promising for biodegradable biomaterials and food applications, but their stability at pH > 2.0 is low. The results presented here show that modified ß-lactoglobulin can also form nanofibrils by heating at neutral pH without prior acidic hydrolysis; the key is removing covalent disulfide bonds via precision fermentation. The aggregation behaviour of various recombinant ß-lactoglobulin variants was systemically studied at pH 3.5 and 7.0. The suppression of intra- and intermolecular disulfide bonds by eliminating one to three out of the five cysteines makes the non-covalent interactions more prevalent and allow for structural rearrangement. This stimulated the linear growth of worm-like aggregates. Full elimination of all five cysteines led to the transformation of worm-like aggregates into actual fibril structures (several hundreds of nanometres long) at pH 7.0. This understanding of the role of cysteine in protein-protein interactions will help to identify proteins and protein modifications to form functional aggregates at neutral pH.
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Amiloide , Lactoglobulinas , Lactoglobulinas/genética , Lactoglobulinas/química , Amiloide/química , Proteínas Amiloidogênicas , Concentração de Íons de Hidrogênio , Dissulfetos/químicaRESUMO
Fungi grow on a great variety of organic and inorganic materials. Colony establishment and growth on solid surfaces require adhesion of spores and hyphae to the substrate, while cell-to-cell interactions among spores and/or hyphae are a prerequisite for the development of three-dimensional mycelial structures such as pellets or biofilms. Surface adherence has been described as a two-step process, comprised of the initial attachment of ungerminated conidia followed by further adhesion of the forming germ tubes and growing hyphae. In the present study, we analyzed the contribution of adhesion of ungerminated spores to pellet and biofilm formation in Aspergillus niger. Mutants deficient in melanin biosynthesis were constructed by the deletion of the alb1 gene, encoding a polyketide synthase essential for pigment biosynthesis. Δalb1 conidia have an altered surface structure and changed physicochemical surface properties. Spore aggregation in liquid culture as well as spore surface attachment differ between the wild type and the mutant in a pH-dependent manner. In liquid culture further pellet formation is unaffected by altered spore-spore interactions, indicating that germ tube and hyphal adherence can compensate for deficiencies in the initial step of spore attachment. In contrast, under conditions promoting adhesion of Δalb1 conidia to polymer surfaces the mutant forms more stable biofilms than the wild type, suggesting that initial spore adhesion supports sessile growth.
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Aspergillus niger/fisiologia , Biofilmes/crescimento & desenvolvimento , Adesão Celular , Esporos Fúngicos/fisiologia , Aspergillus niger/citologia , Aspergillus niger/crescimento & desenvolvimento , Vias Biossintéticas/genética , Deleção de Genes , Hifas/fisiologia , Melaninas/biossíntese , Propriedades de SuperfícieRESUMO
The present study describes the design of bio-pellet morphologies of the industrial working horse Aspergillus niger strains in submerged culture. The novel approach recruits the intended addition of titanate microparticles (TiSiO(4), 8 µm) to the growth medium. As tested for two recombinant strains producing fructofuranosidase and glucoamylase, the enzyme titer by the titanate-enhanced cultures in shake flasks was increased 3.7-fold to 150 U/mL (for fructofuranosidase) and 9.5-fold to 190 U/mL (for glucoamylase) as compared to the control. This could be successfully utilized for improved enzyme production in stirred tank reactors. Stimulated by the particles, the achieved final glucoamylase activity of 1,080 U/mL (fed-batch) and 320 U/mL (batch) was sevenfold higher as compared to the conventional processes. The major reason for the enhanced production was the close association between the titanate particles and the fungal cells. Already below 2.5 g/L the micromaterial was found inside the pellets, including single particles embedded as 50-150 µm particle aggregates in the center resulting in core shell pellets. With increasing titanate levels the pellet size decreased from 1,700 µm (control) to 300 µm. Fluorescence based resolution of GFP expression revealed that the large pellets of the control were only active in a 200 µm surface layer. This matches with the critical penetration depth for nutrients and oxygen typically observed for fungal pellets. The biomass within the titanate derived fungal pellets, however, was completely active. This was due a reduced thickness of the biomass layer via smaller pellets as well as the core shell structure. Moreover, also the created loose inner pellet structure enabled a higher mass transfer and penetration depths for up to 500 µm. The creation of core-shell pellets has not been achieved previously by the addition of microparticles, for example, made of talc or alumina. Due to this, the present work opens further possibilities to use microparticles for tailor-made morphology design of filamentous fungi, especially for pellet based processes which have a long and strong industrial relevance for industrial production.
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Aspergillus niger/metabolismo , Reatores Biológicos , Glucana 1,4-alfa-Glucosidase/metabolismo , Engenharia Metabólica/métodos , beta-Frutofuranosidase/metabolismo , Aspergillus niger/efeitos dos fármacos , Aspergillus niger/fisiologia , Glucana 1,4-alfa-Glucosidase/análise , Proteínas de Fluorescência Verde/metabolismo , Microesferas , Tamanho da Partícula , Silicatos/química , Silicatos/farmacologia , Titânio/química , Titânio/farmacologia , Viscosidade , beta-Frutofuranosidase/análiseRESUMO
The analysis of autofluorescence, often regarded as undesired noise during the imaging of biological samples, allows label free, unbiased detection of NAD(P)H and melanin in native samples. Because both the emission and absorption spectra of these fluorophores overlap and they can hence not be differentiated using emission filters or with different excitation wavelengths, fluorescence lifetime imaging microscopy (FLIM) is used to differentiate between them. In the present paper the application of two-photon excitation microscopy is presented to investigate the autofluorescence of fungal spores. The model organism which was examined is Aspergillus ochraceus. Furthermore a strategy is developed which allows to quantitatively analyze the fluorescence lifetimes of melanin, free NAD(P)H and protein-bound NAD(P)H using forward convolution of a multiexponential decay function with the instrument response function (IRF) and subsequent fitting to the experimental fluorescence data. As a consequence proteins, which are able to bind NAD(P)H, are located with sub-cellular resolution. Furthermore a spatial differentiation of the fluorophores NAD(P)H and melanin inside the spores, is revealed.
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Enzimas/metabolismo , Melaninas/metabolismo , NADP/metabolismo , Espectrometria de Fluorescência/métodos , Aspergillus ochraceus/citologia , Aspergillus ochraceus/metabolismo , Melaninas/química , NADP/químicaRESUMO
Filamentous microorganisms are important work horses in industrial biotechnology and supply enzymes, antibiotics, pharmaceuticals, bulk and fine chemicals. Here we highlight recent findings on the use of microparticles in the cultivation of filamentous bacteria and fungi, with the aim of enabling a more precise control of their morphology towards better production performance. First examples reveal a broad application range of microparticle based processes, since multiple filamentous organisms are controllable in their growth characteristics and respond by enhanced product formation.
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Bactérias/citologia , Biotecnologia/métodos , Fungos/citologia , Microbiologia Industrial/métodos , Microesferas , Bactérias/genética , Bactérias/metabolismo , Fungos/genética , Fungos/metabolismoRESUMO
Microbioreactors (MBRs) have emerged as potent cultivation devices enabling automated small-scale experiments in parallel while enhancing their cost efficiency. The widespread use of MBRs has contributed to recent advances in industrial and pharmaceutical biotechnology, and they have proved to be indispensable tools in the development of many modern bioprocesses. Being predominantly applied in early stage process development, they open up new fields of research and enhance the efficacy of biotechnological product development. Their reduced reaction volume is associated with numerous inherent advantages - particularly the possibility for enabling parallel screening operations that facilitate high-throughput cultivations with reduced sample consumption (or the use of rare and expensive educts). As a result, multiple variables can be examined in a shorter time and with a lower expense. This leads to a simultaneous acceleration of research and process development along with decreased costs.MBRs range from simple miniaturized cultivations vessels (i.e., in the milliliter scale with limited possibilities for process control) to highly complex and automated small-scale microreactors with integrated sensors that allow for comprehensive screenings in very short time or a precise reflection of large-scale cultivation conditions. Progressive developments and improvements in manufacturing and automation techniques are already helping researchers to make use of the advantages that MBRs offer. This overview of current MBR systems surveys the diverse application for microbial and mammalian cell cultivations that have been developed in recent years.
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Reatores Biológicos , Biotecnologia , Animais , Biotecnologia/métodos , MamíferosRESUMO
In the present study, it is shown that the concentration dependency of undefined mixed culture anodic biofilms does not follow a single kinetic curve, such as the Nernst-Monod curve. The biofilms adapt to concentration changes, which inevitably have to be applied to record kinetic curves, resulting in strong shifts of the kinetic parameters. The substrate concentration in a continuously operated bioelectrochemical system was changed rapidly via acetate pulses to record Nernst-Monod curves which are not influenced by biofilm adaptation processes. The values of the maximum current density j max and apparent half-saturation rate constant K s increased from 0.5 to 1 mA cm-2 and from 0.5 to 1.6 mmol L-1, respectively, within approximately 5 h. Double pulse experiments with a starvation phase between the two acetate pulses showed that j max and K s decrease reversibly through an adaptation process when no acetate is available. Pseudo-capacitive charge values estimated from non-turnover cyclic voltammograms (CV) led to the hypothesis that biofilm adaptation and the observed shift of the Nernst-Monod curves occurred due to changes in the concentration of active redox proteins in the biofilm. It is argued that concentration-related parameters of kinetic models for electroactive biofilms are only valid for the operating points where they have been determined and should always be reported with those conditions.