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Diffusion coefficients of actual metabolites in completely active biofilms can be determined by applying a new concept that is based on a constant local activity in the entire biofilm. In that case, a concentration step will be transmitted unattenuated. Subsequently, the diffusion coefficient can be calculated from the response monitored with a microelectrode positioned in the biofilm without quantitative knowledge of the local microbial kinetics. The conditions required for such a constant microbial biofilm activity were formulated in terms of the Thiele modulus and the substrate concentration in the bulk liquid. This proposed method was successfully applied to determine diffusion coefficients of oxygen and glucose in agar gels containing various fractions of active immobilized microorganisms. The values obtained were compared to experimental results from well-defined inert systems. The transient response of oxygen was far more affected by the presence of the immobilized cells than glucose. This can be explained by partition of the diffusing solute between the microbial cells and the aqueous phase.

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 μm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira -like bacteria in situ.

Glucose and pH microprofiles in UASB aggregates were interpreted with respect to the population distribution of fermentative and methanogenic bacteria. In order to map the activity distribution of these bacteria, microprofiles were measured with different substrates (glucose, propionate and acetate) and at varying buffer capacities of the medium. The measurements were compared to calculated microprofiles, in which the spatial location of the activities was varied. The results suggested an inhomogenic activity distribution in the aggregates investigated: fermentatives were located dominantly at the outer 200-300 μm whereas methanogens were homogeneously distributed over the aggregate.

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.

The conversion rate of biological particles is affected by the mass transfer rate in the particle and the surrounding liquid film. The influence of gas production on the external mass transfer rate was investigated in an inorganic model system, consisting of strong acid ion-exchange particles converting a sodium bicarbonate solution. The enhancement by the evolution of carbon dioxide amounted to a factor of 4 compared to sodium hydroxide as a reference. The consequences of the experimental results for biological systems are discussed, and it is concluded that the conversion rate might considerably be influenced under conditions of low turbulence. However, no substantial acceleration by the investigated mechanism is to be expected for suspended biological particles, due to the relatively low gas flux at the surface of the particle.

High operational stability and productivity of co-immobilised systems are important aspects for their successful application in industrial processes. A dynamic model is required to describe artificially co- immobilised systems because the time needed to reach steady state normally exceeds the operational life span of these systems. Time dependent intraparticle concentration profiles and macroscopic conversion were modelled to study the operational stability and productivity of these systems theoretically. The model was used to describe experimental results of ethanol production from maltose by a co-immobilised system of amyloglucosidase and Zymomonas mobilis. Furthermore, the influence of the immobilisation procedure with glutaraldehyde and polyethyleneimine could also be studied with and incorporated in the model. From the model it could be derived that co- immobilised systems performing a consecutive reaction evolve towards a steady state, characterised by a constant concentration of the intermediate in the particle if product inhibition is neglected. Such a situation develops independently of the biomass concentration and the radial position, and has important consequences for co-immobilised systems. When the concentration of the intermediate in the bulk liquid is lower than this constant value in the biocatalyst particle, two regions may be distinguished in the particle: an inactive peripheral region without biomass and an active core with a biomass concentration depending on the substrate and immobilised enzyme concentration. Unlike immobilised single cell systems, it is possible to obtain a real steady state and therefore a stable situation for co- immobilised systems. However, a high operational life time could only be achieved at the expense of the productivity of the biocatalyst particle. A stability criterion is derived which agrees very well with the simulation results.

As an example of the importance and the potential of in situ structure/function studies, nitrifying aggregates from different zones of a lab-scale fluidised bed reactor were analysed by microelectrode measurements of O2, NH4+, NO2−, and NO3− and in situ hybridisation targeting the 16S rRNA of the nitrifying bacteria. A shift from an ammonia oxidising to a nitrite oxidising community is present along the reactor. In the single aggregates an active nitrifying shell of about 100 μm could be related to the maximum abundance of nitrifiers in this zone. Interestingly, the main actors in this system are not representatives of the well-described genera Nitrosomonas and Nitrobacter but some other ammonia oxidisers from the beta subclass of Proteobacteria and a thus far unknown nitrite oxidising population.

Gas bubbles entrapped in biocatalyst particles subjected to hydrostatic pressure oscillations, e.g. during recirculation in loop reactors, will induce intraparticle liquid flows, and thereby enhance mass transfer in excess of diffusion. This ‘breathing particle’ mechanism was demonstrated in methanogenic granules, and led to a typical activity increase of 13% compared with static pressure conditions. From these experimental results and model predictions, it is concluded that convective acceleration of mass transfer in gas-producing systems offers interesting perspectives for the optimisation of biofilm processes in loop reactors. Development of special carrier materials with a central gas-filled cavity could lead to a novel type of bioreactor in which liquid flow through the biocatalyst is decisive.

Publisher Summary This chapter discusses process technology of biotechniques. The number of applications of biological treatment systems for off-gas purification has strongly increased. The application of a biological treatment system is based upon the microbial degradability of the compounds present in the waste gas. The performance of a continuously operating bioreactor is the ultimate result of a complex interaction between the microbiological and physical phenomena, called as the “macrokinetics” of the process. The microkinetics of the degradation process are generally investigated and modeled for pure cultures of suspended microorganism. In many bioreactor systems the degradation also takes place within fixed biofilms, which means that additional mass transfer phenomena (i.e., mass transfer to the biofilm and internal diffusion) should be taken into account. The elimination of organic substrates by microorganism results from the fact that those organisms can use the organic compounds as its sole energy—catabolism, and carbon source—anabolism.

Biofiltration is a technique which is frequently applied for the odour abatement of waste gases. This technique is based on the ability of microorganisms (generally bacteria, and to a small extent moulds and yeasts) to degrade several organic as well as inorganic compounds to mineral end-products, like water and carbon dioxide. In the case of biofiltration, microorganisms are attached to suited packing materials in the filter, which contain the inorganic nutrients necessary for microbial growth. In order to make biofiltration applicable on a larger scale in process industry, it is necessary to find microorganisms able to eliminate compounds which are strange to life, the so-called xenobiotics. At the Eindhoven University of Technology microorganisms were isolated for the elimination of a number of xenobiotics, e.g. aromatic compounds and chlorinated hydrocarbons.

Since the early sixties biological processes have been introduced as a technique for odour abatement of waste gases. Nowadays there is a clear trend to use these systems more broadly. In the last four years these processes have been developed into a technique of increasing importance for air pollution control. These reliable and cheap techniques have proved to be very appropriate for the prevention of air contamination with undesirable components in general.

In the last ten years biotechnological methods have increasingly been used in the treatment of polluted air. The principles of this technique of biofiltration are based on the aerobic conversion of pollutants by microorganisms. At the outset biofiltration was mainly used for odour abatement in waste water treatment plants. Nowadays there is a clear trend to apply biofiltration on a much broader scale. One of the most interesting applications is the degradation of xenobiotic compounds. DHV Consulting Engineers, Eindhoven University of Technology and Clairtech BV have developed the so-called Bioton (R), a modern application of biofiltration. The main advantages of the Bioton are the high possible load, the relatively small space requirements and the large applicability because of the use of specially isolated strains of micro-organisms. Investigations were carried out at a laboratory as well as a pilot-plant scale regarding the degradation of solvents in general.

granules suspended ina mineral mediumlacking glucose exhibited a distinct internal pHdecrease ofabout1U within thegranule, suggesting strong metabolism bytheacidogenic bacteria. Molasses-acclimatized andagedgranules suspended inmineral mediumdidnotexhibit such apHdecrease, suggesting theimportance ofthemetabolic state ofthese acidogens. ThepHgradient didnotoccurindeactivated granules andwas notobservable in strongly buffered media(mineral mediumcontaining 33mM phosphate or reactor liquid). Whenglucose (0.5 to5.0mM) was addedtothemineral medium, granules exhibited a convexpHprofile. Glucose consumption was located exclusively intheouter200to300 .moftheaggregates (meandiameter = 1.5mm).Theaddition of20mM 2-bromoethanesulfonic acidtothemineral mediumindicated that thehigher pHlevels inthecentre ofthegranule appeared toberelated totheactivity ofmethanogens. Itissuggested thatacidogenic activity occurspredominantly intheouter200to300,umoftheaggregate andmethanogenic activity occurs predominantly inthecenter oftheinvestigated granules. Themicrobial community involved inanaerobic digestion processes innatural habitats aswellasinmanufactured digestion systems isknowntobequite complex, comprising hydrolytic, fermentative, acidogenic, andmethanogenic bacteria. Complete anaerobic degradation oforganic matter to methane (CH4)andcarbon dioxide (CO2) requires theconcerted action ofthese different microbial species (4,13,63). Anaerobic wastewater treatment relies onthecontrol of these microbial interactions byapplying appropriate reactor conditions andreactor configurations (22, 40). Theupflow anaerobic sludge blanket (UASB)reactor concept (29, 30) uncouples solid andhydraulic retention times bybiomass accumulation incompact, well-settling aggregates. This granular configuration hasseveral engineering advantages suchasamaximal microorganism-to-space ratio andfacili