From three-dimensional morphology to effective diffusivity in filamentous fungal pellets

Filamentous fungi are exploited as cell factories in biotechnology for the production of proteins, organic acids, and natural products. Hereby, fungal macromorphologies adopted during submerged cultivations in bioreactors strongly impact the productivity. In particular, fungal pellets are known to limit the diffusivity of oxygen, substrates, and products. To investigate the spatial distribution of substances inside fungal pellets, the diffusive mass transport must be locally resolved. In this study, we present a new approach to obtain the effective diffusivity in a fungal pellet based on its three-dimensional morphology. Freeze-dried Aspergillus niger pellets were studied by X-ray microcomputed tomography, and the results were reconstructed to obtain three-dimensional images. After processing these images, representative cubes of the pellets were subjected to diffusion computations. The effective diffusion factor and the tortuosity of each cube were calculated using the software GeoDict. Afterwards, the effective diffusion factor was correlated with the amount of hyphal material inside the cubes (hyphal fraction). The obtained correlation between the effective diffusion factor and hyphal fraction shows a large deviation from the correlations reported in the literature so far, giving new and more accurate insights. This knowledge can be used for morphological optimization of filamentous pellets to increase the yield of biotechnological processes.     

Oxygen diffusion and microbial activity in the composting of dehydrated sewage sludge cakes

The effective diffusivity for oxygen within sewage sludge cakes was measured, and in turn used for the study of microbial activity in the sludge cake composting. The diffusivity was found to be proportional to the one and a half power of the sludge cake porosity. Composting experiments were done using two types of sludge cakes, 5 mesh sieved and pelletized. It was found that the microbial activity in the inner core region of the pelletized sludge cake was significantly less than that in the outer region, even through the rate of oxygen diffusion was fast enough to support the microorganisms' activities. It appears, therefore, that microorganisms acting to degrade organic matter grow primarily on the macroscopic interface between solids and air.     

Perfusion cultures and modelling of oxygen uptake with three-dimensional chondrocyte pellets

Chondrocyte pellets were cultivated in a perfused flow chamber and supplied with medium by a constant flow rate from a conditioning vessel. In this conditioning vessel the medium was aerated and used medium was exchanged semi-continuously. The higher amount of DNA and glycosaminoglycane (GAG) in these pellets compared to control cultures under stationary conditions showed a positive effect of the reactor system, compared to standard culture conditions. A diffusion reaction model was applied to calculate the oxygen uptake of the cell pellet and to describe the oxygen profile within the pellet. The model included diffusion within the cell pellet and oxygen uptake of the cells. Calculated data were compared to experimental data obtained by tissue engineered chondrocyte cell pellets. Model calculations agreed rather well with experimental data.     

Characterization of tissue scaffolds for time-dependent biotransport criteria – a novel computational procedure

This study aims to establish a new computational framework that allows modeling transient oxygen diffusion in tissue scaffolds more efficiently. It has been well known that the survival of cells strongly relies on continuous oxygen/nutrient supply and metabolite removal. With optimal design in scaffold architecture, its ability to sustain long distance oxygen supply could be improved considerably. In this study, finite element based homogenization procedure is first used to characterize the initial effective biotransport properties in silico. These initial properties are proper indicators to prediction of the on-going performance of tissue scaffolds over time. The transient model by adopting an edge-based smoothed finite element method with combination of mass-redistributed method is then established to more efficiently simulate the transient oxygen transfer process in tissue scaffolds. The proposed new method allows large time steps to model the oxygen diffusion process without losing numerical accuracy, thereby enhancing the computational efficiency significantly, in particular for the design optimization problems which typically require numerous analysis iterations. A number of different scaffold designs are examined either under net diffusion without cell seeding, or under cellular oxygen/nutrient uptake with or without considering cell viability. The association between the homogenized effective diffusivity of net scaffold microstructures and corresponding transient diffusion and time-dependent cellular activities is divulged. This study provides some insights into scaffold design.     

Diffusivity of oxygen in aerobic granules

This work for the first time estimated apparent oxygen diffusivity (D(app)) of two types of aerobic granules, acetate-fed and phenol-fed, by probing the dissolved oxygen (DO) level at the granule center with a sudden change in the DO of the bulk liquid. With a high enough flow velocity across the granule to minimize the effects of external mass transfer resistance, the diffusivity coefficients of the two types of granules were estimated with reference to a one-dimensional diffusion model. The carbon source has a considerable effect on the granule diameter (d) and the oxygen diffusivity. The diffusivity coefficients were noted 1.24-2.28 x 10(-9) m2/s of 1.28-2.50 mm acetate-fed granules, and 2.50-7.65 x 10(-10) m2/s of 0.42-0.78 mm phenol-fed granules. Oxygen diffusivity declined with decreasing granule diameter, in particular, the diffusivity of acetate-fed granules is proportional to the size, whereas the diffusivity of phenol-fed granules is proportional to the square of granule diameter. The existence of large pores in granule, evidenced by FISH-CLSM imaging, was proposed to correspond to the noted size-dependent oxygen diffusivity. The phenol-fed granules exhibited a higher excellular polymer (ECP) content than the acetate-fed granules, hence yielding a lower oxygen diffusivity.     

Oxygen and the Spatial Structure of Microbial Communities

Oxygen has two faces. On one side it is the terminal electron acceptor of aerobic respiration – the most efficient engine of energy metabolism. On the other hand, oxygen is toxic because the reduction of molecular O₂ creates reactive oxygen species such as the superoxide anion, peroxide, and the hydroxyl radical. Probably most prokaryotes, and virtually all eukaryotes, depend on oxygen respiration, and we show that the ambiguous relation to oxygen is both an evolutionary force and a dominating factor driving functional interactions and the spatial structure of microbial communities.We focus on microbial communities that are specialised for life in concentration gradients of oxygen, where they acquire the full panoply of specific requirements from limited ranges of PO₂ , which also support the spatial organisation of microbial communities. Marine and lake sediments provide examples of steep O₂ gradients, which arise because consumption or production of oxygen exceeds transport rates of molecular diffusion. Deep lakes undergo thermal stratification in warm waters, resulting in seasonal anaerobiosis below the thermocline, and lakes with a permanent pycnocline often have permanent anoxic deep water. The oxycline is here biologically similar to sediments, and it harbours similar microbial biota, the main difference being the spatial scale. In sediments, transport is dominated by molecular diffusion, and in the water column, turbulent mixing dominates vertical transport.Cell size determines the minimum requirement of aerobic organisms. For bacteria (and mitochondria), the half‐saturation constant for oxygen uptake ranges within 0.05 – 0.1% atmospheric saturation; for the amoeba Acanthamoeba castellanii it is 0.2%, and for two ciliate species measuring around 150 μm, it is 1‐2 % atmospheric saturation. Protection against O₂ toxicity has an energetic cost that increases with increasing ambient O₂ tension. Oxygen sensing seems universal in aquatic organisms. Many aspects of oxygen sensing are incompletely understood, but the mechanisms seem to be evolutionarily conserved. A simple method of studying oxygen preference in microbes is to identify the preferred oxygen tension accumulating in O₂ gradients. Microorganisms cannot sense the direction of a chemical gradient directly, so they use other devices to orient themselves. Different mechanisms in different prokaryotic and eukaryotic microbes are described. In O₂ gradients, many bacteria and protozoa are vertically distributed according to oxygen tension and they show a very limited range of preferred PO₂. In some pigmented protists the required PO₂ is contingent on light due to photochemically generated reactive oxygen species. In protists that harbour endosymbiotic phototrophs, orientation towards light is mediated through the oxygen production of their photosynthetic symbionts. Oxygen plays a similar role for the distribution of small metazoans (meiofauna) in sediments, but there is little experimental evidence for this. Thus the oxygenated sediments surrounding ventilated animal burrows provide a special habitat for metazoan meiofauna as well as unicellular organisms.     

A novel technique for measuring solute diffusivities in entrapment matrices used in immobilization

A novel technique has been developed for measuring effective solute diffusivities in entrapment matrices used for cell immobilization. In this technique radiotracers were used to measure effective diffusivities and equilibrium partition coefficients of the solute between the liquid and solid matrix. Ca-alginate was used in this study, because it is one of the most commonly employed matrices for the immobilization of microbial, plant and mammalian cells. The experimental apparatus consisted of a single spherical Ca-alginate bead which was attached to a rotating rod and immersed in water containing C(14)-glucose. The rotational speed of the spherical bead was controlled and resulted in excellent mixing, and negligible external film mass transfer resistance, which allowed the measurement of true effective solute diffusivity within the solid matrix. The rates of C(14)-glucose diffusion within the Ca-alginate sphere were measured using a scintillation spectrometer. A mathematical model of unsteady-state diffusion in a sphere was used with appropriate boundary conditions, and the effective diffusivity of glucose was found from the best fit of the experimental data using a computer regression analysis method. Using 2% (w/v) Ca-alginate beads in this new radiotracer technique the effective diffusivity and partition coefficient of glucose were found to be 6.62 x 10(-10) m(2)/s and 0.98, respectively. The accuracy, advantages, and simplicity of this new method for diffusivity measurements are also compared to other existing methods.     

Oxygen transfer and culture characteristics of self-immobilized Solanum aviculare aggregates

Oxygen transfer characteristics of self-immobilized Solanum aviculare cells were measured using aggregates 3.0 to 12.5 mm in diameter. Apparent specific oxygen uptake rates in the absence of external boundary layers varied from 5.9 x 10(-11) to 8.5 x 10(-7) kg kg(-1) s(-1) dry weight, but did not decline continuously with increasing particle size. The effective diffusivity of oxygen in deactivated aggregates increased with particle diameter, varying from 5.0 x 10(-11) to 1.0 x 10(-9) m(2) s(-1) or between 2% and 40% of the molecular diffusivity in water at the same temperature. Gas spaces detected in the larger aggregates were confined to the central core and were not distributed throughout the tissue to facilitate oxygen transfer. Oxygen consumption rates in the absence of diffusional limitations were estimated using the relationship between the observable Thiele modulus and effectiveness factor for zero-order reaction. The calculated results indicated severe oxygen limitations in the aggregates, but were inconsistent with the observation that relatively large S. aviculare aggregates contained a high fraction of viable cells and were capable ofactive growth and steroidal alkaloid synthesis. This work suggests that oxygen delivery is facilitated in living plant cell aggregates by mechanisms which depend on metabolic activity and which do not function in deactivated cells. (c) 1995 John Wiley & Sons Inc.     

优化黑曲霉菌丝球形成条件提高豆制品废水处理效率

豆制品废水的处理是豆制品厂的一个重要环节。利用黑曲霉在培养过程中形成的菌丝球处理豆制品废水具有安全性好、菌体回收简单、成本低等优点。本实验中以察氏培养基为基础研究了黑曲霉菌丝球的形成条件,并评价了对豆制品废水的处理效果。结果表明在黑曲霉孢子浓度为7.23×10~3/L~8.68×10~6/L、初始pH 3.5~pH 7.5、葡萄糖浓度为0 g/L~30 g/L的条件下都可以形成茵丝球。在豆制品废水中黑曲霉孢子浓度为1.45×10~4/L~7.23×10~5/L、pH 3.7~pH 6.6均可以形成茵丝球,而且在初始pH 5.6时经过72 h的培养豆制品废水变得澄清,COD下降了56.34%。本研究表明利用黑曲霉在豆制品废水中形成菌丝球的方式是一种有潜力的处理方式。     

Influence of age and structure of Pencillium chrysogenum pellets on the internal concentration profiles

Pellets of Penicillium chrysogenum which were spontaneously formed after a certain stage of a batch fermentation, displayed a considerable structural change in course of their lifetime. Microelectrode studies showed the internal mass transport properties of these pellets (diameter 1–3 mm) to be highly effected by their morphological structure. Relatively young pellets, in an early stage of the batch fermentation, possessed a homogeneous and dense structure. These pellets were only partly penetrated by oxygen (ca. 70 μm) at air saturated bulk conditions. Older pellets, in a final stage of the batch fermentation, were stratified and fluffy. They were completely penetrated by oxygen due to a decreased activity and a higher diffusivity. Investigations with glucose microelectrodes revealed that glucose consumption inside pellets of all lifetimes exclusively occurred in the periphery, indicating that growth was restricted to these regions only.     

Fungal Activity Associated with Decomposing Wood is Affected by Nitrogen Concentration in Water

We examined the effect of two contrasting nitrogen concentrations in water (0.16 and 0.82 mg L^–1) in the mass loss of pre‐conditioned balsa wood, and associated fungal activity, in a laboratory microcosm experiment. Contrary to our predictions given the poor nutrient quality of balsa wood, an increase in dissolved nitrogen concentration did not result in increased mass loss or microbial oxygen consumption. Conidial production was stimulated in high, but not in low nitrogen microcosms, although the number of fungal species was similar between both treatments. The percentage contribution of each fungal species to total conidial production was similar at both N concentrations. The results support the notion that reproductive activity of aquatic hyphomycetes is the most sensitive microbial parameter to changes in the environment. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Internal and external mass transfer in biofilms grown at various flow velocities

It appears that biofilms arrange their internal structure according to the flow velocity at which they are grown, which affects the internal mass transfer rate and microbial activity. In biofilms grown at various flow velocities we determined the vertical profiles of the local relative effective diffusivity (termed D(l)) at several locations within each biofilm. From these profiles we calculated the surface-averaged relative effective diffusivity (termed D(sa)) at various distances from the bottom and plotted it against these distances. The D(sa) decreased linearly toward the bottom, forming well-defined profiles that were different for each biofilm. The gradients of these profiles were multiplied by the diffusivity of oxygen, zeta = D(w) dD(sa)/dz, and plotted versus the flow velocity at which each biofilm was grown. The gradients were low at flow velocities below 10 cm/s, reached a maximum at a flow velocity of 10 cm/s, and decreased again at flow velocities exceeding 10 cm/s. The existence of a maximum indicates a possibility that two opposing forces were affecting the slope of the profiles. To explain these observations we hypothesized that biofilms, depending on the flow velocity at which they are grown, arrange their internal architecture to control (1) the nutrient transport rate and (2) the mechanical pliability needed to resist the shear stress of the water flowing past them. It appears that biofilms attempt to satisfy the second goal first, to increase their mechanical strength, and that they do so at the expense of the nutrient transfer rate to deeper layers. This strength increase is associated with an increase in biofilm density, which slows down the internal mass transport rate. Biofilms grown at low flow velocities exhibit low density and high effective diffusivity but cannot resist higher shear stress, whereas biofilms grown at higher flow velocities are denser and can resist higher shear stress but have a lower effective diffusivity.     

Microflora from leaf debris is suitable for treatment of starch industry wastewater

Biological treatment of industrial waste is a widely practiced technique that generates comparatively less environmentally hazardous waste than other chemical treatment processes. Wet milling of maize generates huge amount of wastewater (5 m³/ton) of low pH with organic matter and nutrients. Anaerobic methanogenic and aerobic bacteria are mostly highly sensitive to low pH. The treatment of wastewater causes huge cost of chemical neutralization or hydraulic recirculation for maintaining neutral pH. In the present study, different microbial consortia isolated from cow dung, active sludge from an anaerobic reactor for treatment of industrial wastewater, and leaf debris from benthic soil were screened for tolerance against low pH and for potential of chemical oxygen demand (COD) removal in order to find out an alternative microbial population for industrial water treatment at low pH. The most effective consortia found from leaf debris were further investigated for optimal operation. The microscopic analysis of leaf debris sludge showed abundance of Gram‐negative methanococci, which was found tolerant to low pH in plate culture method. On further investigation for COD removal from starch industry effluent, they were found to be most effective at pH 5 with highest COD removal rate of 70% and lowest biomass generation of 81%. Hence, it was concluded that the low pH‐tolerant methanogen bacteria, enriched from leaf debris sludge, is highly beneficial for anaerobic treatment of wastewater from several industries including corn starch industry by reducing cost of operation for neutralization to neutral pH and through reducing excess waste sludge production by the treatment system.     

Membrane-aerated microbioreactor for high-throughput bioprocessing

A microbioreactor with a volume of microliters is fabricated out of poly(dimethylsiloxane) (PDMS) and glass. Aeration of microbial cultures is through a gas-permeable PDMS membrane. Sensors are integrated for on-line measurement of optical density (OD), dissolved oxygen (DO), and pH. All three parameter measurements are based on optical methods. Optical density is monitored via transmittance measurements through the well of the microbioreactor while dissolved oxygen and pH are measured using fluorescence lifetime-based sensors incorporated into the body of the microbioreactor. Bacterial fermentations carried out in the microbioreactor under well-defined conditions are compared to results obtained in a 500-mL bench-scale bioreactor. It is shown that the behavior of the bacteria in the microbioreactor is similar to that in the larger bioreactor. This similarity includes growth kinetics, dissolved oxygen profile within the vessel over time, pH profile over time, final number of cells, and cell morphology. Results from off-line analysis of the medium to examine organic acid production and substrate utilization are presented. By changing the gaseous environmental conditions, it is demonstrated that oxygen levels within the microbioreactor can be manipulated. Furthermore, it is demonstrated that the sensitivity and reproducibility of the microbioreactor system are such that statistically significant differences in the time evolution of the OD, DO, and pH can be used to distinguish between different physiological states. Finally, modeling of the transient oxygen transfer within the microbioreactor based on observed and predicted growth kinetics is used to quantitatively characterize oxygen depletion in the system.     

Contrasting resistance and resilience to light variation of the coupled oxic and anoxic components of an experimental microbial ecosystem

Understanding how microbial communities of aquatic ecosystems respond to environmental change remains a critical challenge in microbial ecology. In this study, we used light‐dependent oxic–anoxic micro‐ecosystems to understand how the functioning and diversity of aerobic and anaerobic lake analog communities are affected by a pulse light deprivation. Continuous measurements of oxygen concentration were made and a time series of full‐length 16S rRNA sequencing was used to quantify changes in alpha‐ and beta diversity. In the upper oxic layer, oxygen concentration decreased significantly under light reduction, but showed resilience in daily mean, minimum, and maximum after light conditions were restored to control level. Only the amplitude of diurnal fluctuations in oxygen concentrations did not recover fully, and instead tended to remain lower in treated ecosystems. Alpha diversity of the upper oxic layer communities showed a delayed increase after light conditions were restored, and was not resilient in the longer term. In contrast, alpha diversity of the anoxic lower layer communities increased during the light reduction, but was resilient in the longer term. Community composition changed significantly during light reduction, and showed resilience in the oxic layer and lack of resilience in the anoxic layer. Alpha diversity and the amplitude of daily oxygen fluctuations within and among treatments were strongly correlated, suggesting that higher diversity could lead to less variable oxygen concentrations, or vice versa. Our experiment showed that light deprivation induces multifaceted responses of community function (oxygen respiration) and structure, hence focusing on a single stability component could potentially be misleading.     

Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation

The effects of dissolved oxygen tension and mechanical forces on fungal morphology were both studied in the submerged fermentation of Aspergillus awamori. Pellet size, the hairy length of pellets, and the free filamentous mycelial fraction in the total biomass were found to be a function of the mechanical force intensity and to be independent of the dissolved oxygen tension provided that the dissolved oxygen tension was neither too low (5%) nor too high (330%). When the dissolved oxygen concentration was close to the saturation concentration corresponding to pure oxygen gas, A. awamori formed denser pellets and the free filamentous mycelial fraction was almost zero for a power input of about 1 W/kg. In the case of very low dissolved oxygen tension, the pellets were rather weak and fluffy so that they showed a very different appearance. The amount of biomass per pellet surface area appeared to be affected only by the dissolved oxygen tension and was proportional to the average dissolved oxygen tension to the power of 0.33. From this it was concluded that molecular diffusion was the dominant mechanism for oxygen transfer in the pellets and that convection and turbulent flow in the pellets were negligible in submerged fermentations. The biomass per wet pellet volume increased with the dissolved oxygen tension and decreased with the size of the pellets. This means that the smaller pellets formed under a higher dissolved oxygen tension had a higher intrinsic strength. Correspondingly, the porosity of the pellets was a function of the dissolved oxygen tension and the size of pellets. Within the studied range, the void fraction in the pellets was high and always much more than 50%.     

Conditions de digestion et activité de quelques polysaccharidases dans le tube digestif de l'oursin Paracentrotus lividus (Echinodermata)

The pH level of the gut fluid of the echinoid P. lividus is slightly acidic and its oxygen content is low (from 5 to 8%). An amylase and a cellulase-like enzyme are secreted by the echinoid stomach. Amylolytic activities have been detected also in food pellets from the stomach and from the posterior part of the intestine. This activity of intestinal pellets could be of microbial origin.     

Electrochemical checking of aerobic isolates from electrochemically active biofilms formed in compost

Aims:  To design a cyclic voltammetry (CV) procedure to check the electrochemical activity of bacterial isolates that may explain the electrochemical properties of biofilms formed in compost.
Methods and Results:  Bacteria catalysing acetate oxidation in garden compost were able to form electrochemically active biofilms by transferring electrons to an electrode under chronoamperometry. They were recovered from the electrode surface and identification of the isolates using 16S rRNA sequencing showed that most of them were Gammaproteobacteria, mainly related to Enterobacter and Pseudomonas spp. A CV procedure was designed to check the electrochemical activity of both groups of isolates. Preliminary CVs suggested that the bacteria were not responsible for the catalysis of acetate oxidation. In contrast, both groups of isolates were found to catalyse the electrochemical reduction of oxygen under experimental conditions that favoured adsorption of the microbial cells on the electrode surface.
Conclusions:  Members of the genera Enterobacter and Pseudomonas were found to be able to catalyse the electrochemical reduction of oxygen.
Significance and Impact of the Study:  This study has shown the unexpected efficiency of Enterobacter and Pseudomonas spp. in catalysing the reduction of oxygen, suggesting a possible involvement of these species in biocorrosion, or possible application of these strains in designing bio-cathode for microbial fuel cells.     

Closure effects on oxygen transfer and aerobic growth in shake flasks

Oxygen mass transfer in shake flasks is an important aspect limiting the culture of aerobic microorganisms. In this work, mass transfer of oxygen through a closure and headspace of shake flasks is investigated. New equations for prediction of kGa in shake flasks with closures are introduced. Using Pseudomonas putida, microbial growth on glucose (fast metabolism) and phenol (slow metabolism) in shake flasks with closures were studied, considering both substrate and oxygen restrictions. A combined model for oxygen mass transfer and microbial growth is shown to accurately predict experimental oxygen concentrations and oxygen yield factors during growth experiments more accurately than previous models.