Effective diffusivities and mass fluxes in fungal biopellets.

Mass transport within biological aggregates is a key process that can determine overall turnover rates in submerged cultivations. A parameter commonly used for its description is the effective diffusion coefficient D(eff), which is highly dependent on biomass density and structure. Different approaches have been used to estimate or measure D(eff), yet the data still shows broad scattering. This study provides experimental data on effective diffusivities of oxygen within fungal pellets. A correlation is found with the hyphal gradient (dh/dr), which is a morphological parameter describing the structure of the pellet periphery. Furthermore, the dependency of D(eff) on fluid dynamic conditions at the pellet is investigated. The comparison of the results with data from literature clearly demonstrates the influence of the experimental methodology applied for determination of D(eff). Moreover, it is shown that while diffusion limitation of whole pellets is mainly a function of size, the influence of advection in the outer zone of pellets that is supplied with oxygen is actually rather high. Thus, it is concluded that the effective diffusion coefficient might not be sufficient for the description of mass transport within the pellet periphery for a broad range of realistic fluid dynamic conditions during cultivation. Nevertheless, although actual mass transport rates inside pellets are unknown, mass fluxes can be calculated on the basis of spatially resolved data of oxygen and biomass distribution within the pellet.

Population balance modeling of the conidial aggregation of Aspergillus niger.

Numerous biotechnological production processes are based on the submerse cultivation of filamentous fungi. Process design, however, is often hampered by the complex growth pattern of these organisms. In the morphologic development of coagulating filamentous fungi, like Aspergillus niger, conidial aggregation is the first step of filamentous morphogenesis. For a proper description of this phenomenon it is necessary to characterize conidial populations. Kinetic studies performed with an in-line particle size analyzer suggested that two distinct aggregation steps have to be considered. The first step of conidial aggregation starts immediately after inoculation. Both the rate constants of formation and disintegration of aggregates have been determined by measuring the concentration of conidia at the beginning of the cultivation and the concentration of particles at steady state during the first hours of cultivation. In contrast to the first aggregation step, where the collision of conidia is presumed to be responsible for the process, the second aggregation step is thought to be initiated by germination of conidia. Growing hyphae provide additional surface for the attachment of non- germinated conidia, which leads to a strong decrease in particle concentration. The specific hyphal length growth rate and the ratio of particle concentration to the growing adhesion hyphal surface are decisive matters of the second aggregation step. Both aggregation steps can be described by population dynamics and simulated using the program package PARSIVAL (PARticle SIze eVALution) for the treatment of general particle population balances.

Selection of reference genes for normalisation of specific gene quantification data of Aspergillus niger.

Aspergillus niger is a widely used expression host for homologous and heterologous protein production in biotechnological processes. In order to increase product yields, a thorough optimisation of these cultivation processes is necessary. Considering mRNA as the key molecule, which transports the genetic information between DNA and protein production side, the quantification of product specific gene expression provides useful information about product formation already on the level of transcription. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a powerful tool to obtain data about gene transcription. However, using this technique the choice of an appropriate reference system is a crucial aspect to provide optimal data normalisation. A prominent approach is the use of so called housekeeping genes as internal references. However, validation of the usability of these reference genes is the fundamental step before starting with qRT-PCR experiments. Adequate reference genes for A. niger have not been published so far. Therefore, 10 possible candidate genes from different functional classes were selected and their applicability as internal references validated. Transcript levels of these genes were compared in sets of 9, 41 and 19 samples from diverse cultivations of A. niger. Under the chosen experimental conditions, the genes act, sarA and cox5 have been identified as genes with the most stable gene expression. The three reference genes were used to normalise qRT-PCR data for glaA gene expression which showed a high correlation with glucoamylase production in continuous cultivations.

Modelling and experimental investigation of environmental influences on the acetate and methane formation in solid waste.

An anaerobic reaction model is represented and used for simulation of the biodegradation of organic compounds and the generation of biogas. The model is based on fundamental relationships among physical, chemical, thermodynamic and microbial processes occurring in municipal landfills. Local microbially mediated degradation processes occurring in municipal landfills are simulated in terms of hydrolysis of readily and inherently degradable organic matter, the formation of acetate as surrogate for intermediary low-molecular carbon substrates, and the generation of the biogases CH4 and CO2. Thus, the overall decomposition of the organic matter has been assumed to follow three sequential anaerobic reactions steps: hydrolysis, acetogenesis and methanogenesis. In order to study the impact of environmental factors on the biological decomposition processes, experiments have been conducted to investigate the effect of temperature and water content. In the degradation model, the impact of temperature and water content was defined as reaction rate influencing factors. Further, waste samples have been taken from four drill holes on a municipal landfill near Wolfsburg (Germany) and used to analyze and to describe the waste composition and prevailing environmental conditions dependent on the depth of the drill hole. The data and waste samples obtained from the landfill have also been used for model development and validation.

Treatment of dyehouse liquors in a biological sequencing batch reactor with recursive chemical oxidation.

A new developed sequencing batch process for the purification of residual water containing concentrated azo dye was investigated. Within a treatment cycle the biological anoxic decolorization, followed by an aerobic mineralization of organic metabolites in combination with the biodegradability-achieving partial oxidation with ozone are carried out sequentially. The split flow can be destructively purified to 90% with respect to the parameter DOC. It was decolorized to an extent of 98% and the toxicity measured by the bioluminescence test decreased up to 99%. With an unspecific facultative anaerobic bacterial mixed culture anoxic decolorization of the residual liquor (20 gdye/L) without addition of an external auxiliary substrate was observed. In the first phase of the treatment cycle, the azo dye-molecules are cleft at the azo bond by biochemical reduction which leads to the corresponding sulfonated aromatic amines. In the following aerobic phase the cleft products were mineralized by the same microorganisms in the same reactor. Because of the recalcitrant and respectively toxic character of a part of the remaining metabolites, further aerobic mineralization was initialized by partial oxidation with ozone. The recursive ozonization in a recircled stream with biological post-treatment of the transformed substances led to an increased reaction selectivity and lower consumption of ozone. The results have shown that the chosen sequencing batch reactor with the ozonization bypass is suitable for an effective treatment of high concentrated dyehouse liquors.

Simulation of biofilm growth, substrate conversion and mass transfer under different hydrodynamic conditions.

The hydrodynamic conditions and the substrate load in biofilm systems are two main parameters which influence the biofilm growth in particular the structure, density and thickness. In a long term study on heterotrophic biofilms in biofilm tube reactors the investigation has focussed on mass transfer at the bulk/biofilm interface, the biofilm density and the substrate conversion rates. To study the mass transfer phenomena at the bulk/biofilm interface oxygen profiles have been measured directly in tube reactors with microelectrodes. Microelectrode studies, substrate conversion rates and biofilm densities were used to formulate model equations for the simulation of biofilm growth under different hydrodynamic and substrate conditions. It can be shown that the mass transfer at any time is strongly coupled with the growth conditions during the biofilm cultivation. On the one side the calculated Sherwood numbers were coupled to the present hydraulic conditions, on the other side in addition the growth conditions such as growth rate and Reynolds number during biofilm cultivation were considered.

Behaviour of biofilm systems under varying hydrodynamic conditions.

Heterotrophic biofilms were cultivated in long-term experiments in biofilm tube reactors. During the biofilm cultivation the substrate loading of glucose was kept constant while the hydrodynamic conditions were changed stepwise. To describe the behaviour of the biofilm structure under these varying flow conditions the mass transfer and transport at the bulk/biofilm interface and inside the biofilm was investigated with oxygen microelectrodes. Furthermore, the biofilm density was used to describe the biofilm compactness before and after the change of the hydrodynamic condition. The obtained results show that the biofilm density and also the substrate flux decreased with decreasing flow velocity in the bulk phase. Additionally the slope of the oxygen concentration profiles decreased and the thickness of the concentration boundary layer increased. On the other hand, increasing the flow velocity in the bulk phase led both to a higher biofilm density and a higher maximum substrate flux. The biofilm surface became more homogenous and the thickness of the concentration boundary layer decreased. The time for adaptation of the biofilm structure after changing the hydrodynamic conditions ranged between 1 and 3 weeks.

Growth, structure and oxygen penetration in particle supported autotrophic biofilms.

Particle supported autotrophic biofilms were cultivated in external-loop airlift reactors at two different pumice concentrations. Oxygen microelectrodes were used to investigate substrate transport and conversion. A special flow cell was designed for the measurement of oxygen concentration profiles in the particle supported biofilms under defined hydrodynamic conditions. The oxygen concentration profiles inside the biofilms were found to be steeper at high flow velocities in the bulk phase of the flow cell compared to those at low flow velocities. Furthermore, the oxygen flux increased and the thickness of the concentration boundary layer decreased with increasing flow velocity. This dependence was found to be more pronounced in less dense biofilms out of airlift reactors with lower pumice concentrations. In addition confocal laser scanning microscopy (CLSM) was used to visualize the biofilm structure. The volume fractions of bacteria and extracellular polymeric substances (lectin-specific EPS-glycoconjugates) were measured in living fully hydrated biofilms. Both the microelectrode and CLSM measurement showed the influence of shear stress on particle supported biofilms. A higher particle concentration led to dense biofilms with a homogeneous surface, lower thickness of the concentration boundary layer and steeper oxygen concentration profiles. The combination of both techniques allows a detailed and quantitative characterisation of particle associated biofilm structure and function.

Experimental investigation and modelling of the effect of sulfate on anaerobic biodegradation processes in municipal solid waste.

The experimental investigations have been carried out in two parts. First, the biodegradation of the organic compounds in the municipal solid waste has been investigated with focus on different sulfate concentrations influencing the methane formation. Second, the inhibition effect of hydrogen sulfide in solution (H2S(aq)) on the acetate, and methane formation, respectively, has been studied at different pH and temperature values. In solution the equilibrium of hydrogen sulfide (H2S(aq)) and the hydrogen sulfide anion (HS-(aq)) mainly depends on pH. At pH 6.3 the favoured species is H2S(aq) whereas at pH 7.7 the HS-(aq) species is favoured. Additionally, the experiments have been carried out at two different temperatures (35 degrees C and 55 degrees C). According to the acetate and methane formation the H2S(aq) species is observed to have an inhibitory effect. All simulations based on a biodegradation model represented are in good agreement with the experimental data obtained.

Oxygen profiles and biomass distribution in biopellets of Aspergillus niger.

Morphology of fungal pellets has a significant influence on mass transfer and turnover processes in submerged cultures. There are many reports in literature that biomass is not distributed homogeneously over the pellet radius, yet quantitative data is rare. This study presents a method for the quantification of fungal pellet structure (Aspergillus niger). Confocal laser scanning microscopy (CLSM) is used in combination with image analysis freeware (Image J). Hyphal distribution is resolved spatially in radial direction. Quantitative morphological parameters are derived from digital images especially from the peripheral regions of the pellet that are not oxygen limited. This morphological information is combined with data of microelectrode measurements in the same pellets. Results show that the morphological parameters obtained can describe the impact of pellet structure on oxygen gradients much better than average biomass density. It is concluded that CLSM and image analysis are powerful tools not only to generate valuable data for quantitative description of pellet morphology. In addition, this data may be used in mathematical models to improve predictions of mass transfer and substrate conversion in mycelial aggregates.

Influence of mechanical stress and surface interaction on the aggregation of Aspergillus niger conidia.

Productivity of fungal cultures is closely linked with their morphologic development. Morphogenesis of coagulating filamentous fungi, like Aspergillus niger, starts with aggregation of conidia, also denominated as spores. Several parameters are presumed to control this event, but little is known about their mode of action. Rational process optimization requires models that mirror the underlying reaction mechanisms. An approach in this regard is suggested and supported by experimental data. Aggregation kinetics was examined for the first 15 h of cultivation under different cultivation conditions. Mechanical stress was considered as well as pH-dependent surface interaction. Deliberations were based on a two-step aggregation mechanism. The first aggregation step is only affected by the pH-value, not by the fluid dynamic conditions in the bioreactor. The second aggregation step, in contrast, depends on the pH-value as well as on agitation and aeration induced power input. For the given experimental set-up, agitation had much more influence than aeration. In addition, hyphal growth rate was determined to be the driving force for the second aggregation step.

Morphology and productivity of filamentous fungi.

Cultivation processes involving filamentous fungi have been optimised for decades to obtain high product yields. Several bulk chemicals like citric acid and penicillin are produced this way. A simple adaptation of cultivation parameters for new production processes is not possible though. Models explaining the correlation between process-dependent growth behaviour and productivity are therefore necessary to prevent long-lasting empiric test series. Yet, filamentous growth consists of a complex microscopic differentiation process from conidia to hyphae resulting in various macroscopically visible appearances. Early approaches to model this morphologic development are recapitulated in this review to explain current trends in this area of research. Tailoring morphology by adjusting process parameters is one side of the coin, but an ideal morphology has not even been found. This article reviews several reasons for this fact starting with nutrient supply in a fungal culture and presents recent advances in the investigation of fungal metabolism. It illustrates the challenge to unfold the relationship between morphology and productivity.

Substrate utilization and mass transfer in an autotrophic biofilm system: Experimental results and numerical simulation.

An autotrophic biofilm has been investigated for over 10 months in a biofilm tube reactor. The objective of this investigation was the verification and improvement of a biofilm model. The use of a Clark-type oxygen microelectrode in situ allowed the determination of the substrate flux in the biofilm. Also, the population dynamics of the autotrophic bacteria could be evaluated by varying the substrate conditions. Simulation of the experimental results showed that the liquid phase of the biofilm decreased with biofilm depth. This could be described by a logistic function. The density of the inert volume fraction was found to be higher than that of the viable bacteria. This was verified in a nonsubstrate phase of 5 weeks. Growth and decay of the autotrophic bacteria could be described by the growth, endogenous respiration, and death processes. Mass transfer coefficients at the bulk/biofilm interface were evaluated. They were found to be one order of magnitude higher than those known from hydrodynamics in tubes without a biofilm. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 363-371, 1997.

Biodegradation by immobilized bacteria in an airlift-loop reactor-influence of biofilm diffusion limitation.

Naphthalene-2-sulfonate was degraded by submerse growing Pseudomonads in a chemostat culture. The kinetic parameters for the Monod equation, including Pirts maintenance energy, were calculated from these experiments regarding naphthalene-2-sulfonate as substrate and oxygene as cosubstrate. By immobilizing the bacteria on sand particles, the degradation of naphthalene-2-sulfonate was carried out in a specialy designed three-phase airlift-loop reactor in a completely fluidized state. From these experiments, the influence of biofilm diffusion limitation on reaction kinetics and criteria for stable biofilm formation on sand particles were obtained.

Cellulose degradation and monitoring of viscosity decrease in cultures of Cellulomonas uda grown on printed newspaper.

The rheological behavior of cultures of Cellulomonas uda with shredded printed newspaper as the carbon source was studied. The initial substrate concentrations ranged from 23 to 60 g/L. The changes in apparent viscosity were followed on-line by applying a commercially available process viscometer and discretely using a rotational viscometer with an anchor impeller. During the time of highest cellulose degradation, the broths exhibited a pseudoplastic behavior which could be explained satisfactorily by the power-law model. At the end of cultivation when cellulose degradation slowed down, the broths became Newtonian in behavior. Endo-1,4-beta-glucanase, 1,4-beta-xylanase, beta-glucosidase, and beta-xylosidase activities were also determined during cultivation as well as cellulose degradation and cell mass production. The beginning of endoglucanase formation and the start of the final viscosity decrease of the bacterial paper pulp suspensions could be correlated.

Kinetic studies on the aggregation of Aspergillus niger conidia.

Morphology has a crucial effect on productivity and the supply of substrate for cultures of filamentous fungi. However, cultivation parameters leading to the desired morphology are often chosen empirically as the mechanisms governing the processes involved are usually unknown. For coagulating microorganisms like Aspergillus niger the morphological development is considered to start with the aggregation of conidia right after inoculation. To elucidate the mechanism of this process, kinetic studies were carried out using an in-line particle size analyzer. Based on the data obtained from these experiments a model for conidial aggregation is proposed in this article. It consists of two separate aggregation steps. The first one takes place immediately after inoculation, but only leads to a small decrease of total particle concentration. Most suspended conidia aggregate after a second aggregation step triggered by germination and hyphal growth. Aggregation velocity of this second phase is linearly dependent on the particle growth rate.

Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium.

Under anaerobic conditions the sulfonated azo dye Mordant Yellow 3 was reduced by the biomass of a bacterial consortium grown aerobically with 6-aminonaphthalene-2-sulfonic acid. Stoichiometric amounts of the aromatic amines 6-aminonaphthalene-2-sulfonate and 5-aminosalicylate were generated and excreted into the medium. After re-aeration of the culture, these amines were mineralized by different members of the bacterial culture. Thus, total degradation of a sulfonated azo dye was achieved by using an alternating anaerobic-aerobic treatment. The ability of the mixed bacterial culture to reduce the azo dye was correlated with the presence of strain BN6, which possessed the ability to oxidize various naphthalenesulfonic acids. It is suggested that strain BN6 has a transport system for naphthalenesulfonic acids which also catalyzes uptake of sulfonated azo dyes. These dyes are then gratuitously reduced in the cytoplasm by unspecific reductases.

Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms.

In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing heterotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms.

Modelling of the biodegradation of organic matter in municipal landfills.

A model is presented to simulate the biodegradation of easily and slowly hydrolyzable organic matter, as well as the generation of biogas and heat release. The model is based on fundamental relationships among physical/chemical, thermodynamical and microbial processes occurring in municipal landfills. Local, microbially-mediated degradation processes occurring in municipal landfills, are simulated in terms of the hydrolysis of solid organic matter, the formation of glucose and acetate as intermediary carbon substrates and the generation of the biogases CH4 and CO2. Thus, the overall decomposition of the organic matter has been assumed to follow four sequential biochemical reactions: hydrolysis, acidogenesis, acetogenesis and methanogenesis. In order to study the impact of environmental factors on the biological decomposition processes, pH, temperature and hydrogen changes have been integrated into the degradation model as inhibition terms.