Growth of Artemisia annua hairy roots in liquid- and gas-phase reactors

Artemisia annua hairy roots were grown in liquid-phase bubble column and gas-phase nutrient mist reactors. In most cases the bubble column reactor accumulated more biomass than the mist reactor; the highest final biomass concentrations observed were 15.3 g DW/L in the bubble column reactor and 14.4 g DW/L in the mist reactor. Further analysis showed that the average specific growth rate in the mist reactors was essentially constant and independent of the biomass concentration at the beginning of the mist mode. In contrast, at low packing densities the average growth rate in the bubble column reactors was higher than in the mist reactors, decreasing to comparable rates at high packing densities. Finally, an aerosol deposition model was used to compare the volume of medium captured by the root bed in the mist reactor to the volume of medium required to maintain a specified growth rate. The results suggest that under the current operating conditions, lower growth rates in the mist reactor may be due to insufficient nutrient availability.     

Biomass production of hairy roots of Artemisia annua and Arachis hypogaea in a scaled‐up mist bioreactor

Hairy roots have the potential to produce a variety of valuable small and large molecules. The mist reactor is a gas phase bioreactor that has shown promise for low‐cost culture of hairy roots. Using a newer, disposable culture bag, mist reactor performance was studied with two species, Artemisia annua L. and Arachis hypogaea (peanut), at scales from 1 to 20 L. Both species of hairy roots when grown at 1 L in the mist reactor showed growth rates that surpassed that in shake flasks. From the information gleaned at 1 L, Arachis was scaled further to 4 and then 20 L. Misting duty cycle, culture medium flow rate, and timing of when flow rate was increased were varied. In a mist reactor increasing the misting cycle or increasing the medium flow rate are the two alternatives for increased delivery of liquid nutrients to the root bed. Longer misting cycles beyond 2–3 min were generally deemed detrimental to growth. On the other hand, increasing the medium flow rate to the sonic nozzle especially during the exponential phase of root growth (weeks 2–3) was the most important factor for increasing growth rates and biomass yields in the 20 L reactors. A. hypogaea growth in 1 L reactors was µ = 0.173 day^?1 with biomass yield of 12.75 g DW L^?1. This exceeded that in shake flasks at µ = 0.166 day^?1 and 11.10 g DW L^?1. Best growth rate and biomass yield at 20 L was µ = 0.147 and 7.77 g DW L^?1, which was mainly achieved when medium flow rate delivery was increased. The mist deposition model was further evaluated using this newer reactor design and when the apparent thickness of roots (+hairs) was taken into account, the empirical data correlated with model predictions. Together these results establish the most important conditions to explore for future optimization of the mist bioreactor for culture of hairy roots. Biotechnol. Bioeng. 2010;107: 802–813. © 2010 Wiley Periodicals, Inc.     

Gas–liquid dispersion in a fibrous fixed bed biofilm reactor at growth and non-growth conditions

There is limited data on gas dispersion characteristics of fixed bed biofilm reactors under growth and non-growth conditions. In this paper, the gas–liquid dispersion of a bubble bed packed with a fibrous structured packing for biofilm application is studied. The reactor is operated with Pseudomonas putida aimed at aniline degradation in wastewater. Gas hold-up and bubble size distribution are determined. Running gas–liquid reaction conditions as well as non-reactive flow gas hold-up and bubble size distribution in the presence of surface-active and viscous components were measured. The properties of the gas dispersion proved to be stabilized by the fibrous bed presence and showed improvement of the dispersion parameter by the packing. Gas hold-up was found to increase monotonously with the rise of gas superficial velocity and viscosity and with surface tension fall. Liquid superficial velocity showed marginal effect. Apart from showing high gas hold-up and low bubble size due to surface-active and viscous dissolved elements, the biochemical reaction did not pose any significant additional effect. In agreement with the expected lack of bubble coalescence and break-up in the highly ionic solution practiced, the population size distribution and average bubble size were found to vary with the major operation factors opposite to their gas hold-up contribution. Gas hold-up was correlated with the specific bubble-to-channel size ratio and further with the variables considered. An empirical equation is proposed that relates gas hold-up with all studied variables. Assuming geometric similarity of the prototype and the real vessels, the equation as well as its corresponding range of fluid velocities can be used for bioreactor design and scale-up. The results concerning the gas hold-up are shown to be comparable with previous studies of mesh wire packing.     

Outdoor helical tubular photobioreactors for microalgal production: modeling of fluid-dynamics and mass transfer and assessment of biomass productivity

The production of the microalga Phaeodactylum tricornutum in an outdoor helical reactor was analyzed. First, fluid dynamics, mass-transfer capability, and mixing of the reactor was evaluated at different superficial gas velocities. Performance of the reactor was controlled by power input per culture volume. A maximum liquid velocity of 0.32 m s(-1) and mass transfer coefficient of 0.006 s(-1) were measured at 3200 W m(-3). A model of the influence of superficial gas velocity on the following reactor parameters was proposed: gas hold-up, induced liquid velocity, and mass transfer coefficient, with the accuracy of the model being demonstrated. Second, the influence of superficial gas velocity on the yield of the culture was evaluated in discontinuous and continuous cultures. Mean daily values of culture parameters, including dissolved oxygen, biomass concentration, chlorophyll fluorescence (F(v)/F(m) ratio), growth rate, biomass productivity, and photosynthetic efficiency, were determined. Different growth curves were measured when the superficial gas velocity was modified-the higher the superficial gas velocity, the higher the yield of the system. In continuous mode, biomass productivity increased by 35%, from 1.02 to 1.38 g L(-1) d(-1), when the superficial gas velocity increased from 0.27 to 0.41 m s(-1). Maximal growth rates of 0.068 h(-1), biomass productivities up to 1.4 g L(-1) d(-1), and photosynthetic efficiency of up to 15% were obtained at the higher superficial gas velocity of 0.41 m s(-1). The fluorescence parameter, F(v)/F(m), which reflects the maximal efficiency of PSII photochemistry, showed that the cultures were stressed at average irradiances within the culture higher than 280 microE m(-2) s(-1) at every superficial gas velocity. For nonstressed cultures, the yield of the system was a function of average irradiance inside the culture, with the superficial gas velocity determining this relationship. When superficial gas velocity was increased, higher growth rates, biomass productivities, and photosynthetic efficiencies were obtained for similar average irradiance values. The higher the superficial gas velocity, the higher the liquid velocity, with this increase enhancing the movement of the cells inside the culture. In this way the efficiency of the cells increased and higher biomass concentrations and productivities were reached for the same solar irradiance.     

Quantifying relationships between rooting traits and water uptake under drought in Mediterranean barley and durum wheat

In Mediterranean regions drought is the major factor limiting spring barley and durum wheat grain yields. This study aimed to compare spring barley and durum wheat root and shoot responses to drought and quantify relationships between root traits and water uptake under terminal drought.One spring barley（Hordeum vulgare L. cv. Rum） and two durum wheat Mediterranean cultivars（Triticum turgidum L. var durum cvs Hourani and Karim） were examined in soil‐column experiments under well watered and drought conditions. Root system architecture traits, water uptake, and plant growth were measured. Barley aerial biomass and grain yields were higher than for durum wheat cultivars in well watered conditions. Drought decreased grain yield more for barley（47%） than durum wheat（30%, Hourani）. Root‐to‐shoot dry matter ratio increased for durum wheat under drought but not for barley, and root weight increased for wheat in response todrought but decreased for barley. The critical root length density（RLD） and root volume density（RVD） for 90% available water capture for wheat were similar to（cv. Hourani） or lower than（cv. Karim） for barley depending on wheat cultivar. For both species, RVD accounted for a slightly higher proportion of phenotypic variation in water uptake under drought than RLD.     

Feasibility of expanded granular sludge bed reactors for the anaerobic treatment of low-strength soluble wastewaters

The application of the expanded granular sludge bed (EGSB) reactor for the anaerobic treatment of low-strength soluble wastewaters using ethanol as a model substrate was investigated in laboratory-scale reactors at 30oC. Chemical oxygen demand (COD) removal efficiency was above 80% at organic loading rates up to12 g COD/L . d with influent concentrations as low as 100 to 200 mg COD/L. These results demonstrate the suitability of the EGBS reactor for the anaerobic treatment of low-strength wastewaters. The high treatment performance can be attributed to the intense mixing regime obtained by high hydraulic and organic loads. Good mixing of the bulk liquid phase for the substrate-biomass contact and adequate expansion of the substrate-biomass contact and adequate expansion of the sludge bed for the degassing were obtained when the liquid upflow velocity (V(up)) was greater than 2.5 m/h. Under such conditions, an extremely low apparent K(s) value for acetoclastic methanogenesis of 9.8 mg COD/L was observed. The presence of dissolved oxygen in the wastewater had no detrimental effect on the treatment performance. Sludge piston flotation from pockets of biogas accumulating under the sludge bed occurred at V(up) lower than 2.5 m/h due to poor bed expansion. This problem is expected only in small diameter laboratory-scale reactors. A. more important restriction of the EGSB reactor was the sludge washout occurring at V(up) higher than 5.5 m/h and which was intensified at organic loads higher than 7 g COD/L. d due to buoyancy forces from the gas production. To achieve an equilibrium between the mixing intensity and the sludge hold-up, the operation should be limited to an organic loading rate of 7 g COD/L d. and to a liquid up-flow velocity between 2.5 and 5.5 m/h (c) 1994 John Wiley & Sons, Inc.     

Strategies for penicillin fermentation in tower-loop reactors

Since it has not been possible to produce penicillin in tower-loop reactors with highly viscous filamentous molds of Penicillium chrysogenum which are employed in stirred-tank reactors, a new strategy has been developed to avoid the formation of this morphology and to use the pellet form of the fungi. When employing definite impeller speeds in the subculture in connection with definite inoculum amounts and substrate concentrations in the main culture (bubble column), it is possible to generate a suspension of isolated small pellets, which shows a low broth viscosity up to a sediment content of 45% over the entire fermentation time. Volumetric mass-transfer coefficients k(L)as are by a factor of 4 to 5 higher in these pellet suspensions than in filamentous broths. It was easy to maintain the necessary oxygen supply for penicillin production in these pellet suspensions. Under these conditions the specific penicillin productivities were higher with regard to power input (up to 90%), biomass, and consumed substrate than in the stirred-tank reactors with highly viscous filamentous morphology of the fungi. Under nonoptimized operating conditions the absolute penicillin production in the tower loop was 35% lower than in the stirred-tank reactor due to lower possible biomass concentrations. The separation of the biomass, and therefore the penicillin recovery, is much simpler when employing pellets. It is shown how the particular mass transfer resistances at the gas/liquid and liquid/pellet interfaces and within the pellets change with the pellet diameter. There should be a particular pellet diameter at which penicillin productivity has its maximum. These investigations indicate that the use of tower-loop reactors can, in the future, be an alternative for more economical penicillin production methods.     

Region-dependent oxygen transfer rate in the rectangular airlift reactor

Gas hold-up and the oxygen transfer in the zones of the internal loop airlift reactor with rectangular cross-section was studied. It was found, that the downcomer to the riser gas hold-up ratio depends on the gas flow rate, the physicochemical properties of the system and on the reactor height. The ratio of the downcomer mass transfer coefficient to the global mass transfer coefficient was less than 6%. The ratio of the downcomer to the global mass transfer coefficient slightly increased with increase of the gas flow rate and decreased with increase of the liquid viscosity. The proposed correlation for the global overall mass transfer coefficient predicts the experimental data well within 16.6% deviation. It was confirmed that the reactor height is the important parameter for a design and a scale-up of the airlift reactors.     

Effect of acid irrigation and liming on root growth of Norway spruce

The effect of acid irrigation and liming on fine root growth of Norway spruce (Picea abies [L.] Karst.) was studied in an approximately 80-year-old forest stand in southern Germany (Höglwald). Root growth was measured mainly on root windows and in addition by soil core sampling. Root growth rate showed a typical pattern in the course of a year with a maximum in August. Acid irrigation depressed root growth rate, whereas liming, particularly in combination with acid irrigation, markedly increased root growth in the humic layer and the upper 0–5 cm of the mineral soil. The treatment effects on root growth in the mineral soil below 5 cm were small and not significant. Root growth rate was not correlated with the concentration of aluminium (Al) or the molar ratio of calcium (Ca) to Al in the soil solution. The results suggest that inhibition of root growth by acid irrigation is a direct effect of high proton concentrations in the irrigation water, and that enhancement of root growth by liming is caused by an improved supply of mineral nutrients and higher biological activity.     

Characterization of agitation environments in 250 ml spinner vessel, 3 L,and 20 L reactor vessels used for animal cell microcarrier culture

Three dimensional particle tracking velocimetry (3-D PTV) was used to characterize the flow fields in the impeller region of three microcarrier reactor vessels. Three typical cell culture bioreactors were chosen: 250 ml small-scale spinner vessels, 3 L bench-scale reactor, and 20 L medium-scale reactor. Conditions studied correspond to the actual operating conditions in industrial setting and were determined based on the current scale-up paradigm: the Kolmogorov eddy length criterion. In this paper we present characterization of hydrodynamics on the basis of flow structures produced because of agitation. Flow structures were determined from 3-D mean velocity results obtained using 3-D PTV. Although the impellers used in 3 L and 20 L reactors were almost identical, the flow structures produced in the two reactors differed considerably. Results indicate that near geometric scale up does not necessarily amount to scale-up of flow patterns and indicates that intensity as well as distribution of energy may vary considerably during such a scale-up.     

Startup of anaerobic fluidized bed reactors with acetic acid as the substrate

The startup of anaerobic fluidized bed reactors, which use Manville R-633 beads as the growth support media, acetate enriched bacterial culture as the inoculum, and acetic acid as the sole substrate, is studied. Tow startup strategies are evaluated: one based on maximum and stable substrate utilization and another based on maximum substrate loading controlled by reactor pH. The startup process is characterized using a number of operational parameters.The reactors again excellent total organic carbon (TOC) removal (i.e., > 97% at a feed concentration of 5000 mg TOC/L) and stable methane production (i.e., 0.90 L CH(4)/g TOC, where TOC(r) is TOC removed) at a early stage of the startup process, regardless of the strategies applied. The loading can be increased rapidly without the danger of being overloaded. Significant losses of growth support media and biomass caused by gas effervescence at higher loadings limits the maximum loading that can be safely applied during startup process.A high reactor immobilized biomass inventory is achievable using the porous growth support media (e.g., Manville 633 beads). A rapid increase in loading creates a substrate rich environment that yields more viable reactor biomass. Both substrate utilization rate (batch and continuous) and immobilized biomass inventory stabilize concomitantly at the late stage of the startup process, indicating the attainment of steady-state conditions in reactors. Therefore, they are better parameters that TOC removal and methane production for characterizing the entire startup process of aerobic fluidized bed reactor.The strategy based on maximum substrate loading controlled by reactor pH significantly shortens the startup time. In this case, the reactor attains steady-state conditions approximately 140 days after startup. On the other hand, a startup time of 200 days is required when the strategy based maximum substrate utilization is adopted. (c) 1993 John Wiley & Sons, Inc.     

The effects of elevated CO2 concentrations on the root growth of Lolium perenne and Trifolium repens grown in a FACE* system

Lolium perenne and Trifolium repens were grown in a Free Air CO₂ Enrichment (FACE) system at elevated (600 μimol mol-¹) and ambient (340 μmol mol-¹) carbon dioxide concentrations during a whole growing season. Using a root ingrowth bag technique the extent to which CO₂ enrichment influenced the growth of L, perenne and T. repens roots under two contrasting nutrient regimes was examined. Root ingrowth bags were inserted for a fixed time into the soil in order to trap roots. It was also possible to follow the mortality of roots in bags inserted for different time intervals. Root ingrowth of both L. perenne and T. repens increased under elevated CO₂ conditions. In L. perenne, root ingrowth decreased with increasing nutrient fertilizer level, but for T. repens the root ingrowth was not affected by the nutrient application rate. Besides biomass measurements, root length estimates were made for T, repens. These showed an increase under elevated CO₂ concentrations. Root decomposition appeared to decrease under elevated CO₂ concentrations. A possible explanation for this effect is the observed changes in tissue composition, such as the increase in the carbon: nitrogen ratio in roots of L. perenne at elevated CO₂ concentrations.     

Aerobic oxidation of hydrogen sulfide by Thiobacillus denitrificans

It has been demonstrated that Thiobacillus denitrificans may be readily cultured aerobically in batch and continuous flow reactors on H(2)S(g) under sulfide limiting conditions. Under these conditions sulfide concentrations in the culture medium were less than 1muM resulting in very low concentrations of H(2)S in the reactor outlet gas. Biomass yield under aerobic conditions was much lower than previously reported for anaerobic conditions, presumably because of oxygen inhibition of growth. However, biomass yield was not affected by steady state oxygen concentration in the range of 45muM-150muM. Biomass yield was also observed to be essentially independent of specific growth rate in the range of 0.030-0.053 h(-1). Indicators of reactor upset were determined and recovery from upset conditions demonstrated. Maximum loading of the biomass for H(2)S oxidation under aerobic conditions was observed to be 15.1-20.9 mmol/h/g biomass which is much higher than previously reported for aerobic conditions. Other aspects of the stoichiometry of aerobic H(2)S oxidation are also reported.     

海南岛4个热带阔叶树种前5级细根的形态、解剖结构和组织碳氮含量

细根具有复杂的分支系统, 以根序(root order)为取样单元的细根生理生态学研究正在成为根系生态学研究领域的重要内容。该研究以海南岛尖峰岭4个热带阔叶树种海南蕈树(Altingia obovata)、厚壳桂(Cryptocarya chinensis)、山杜英(Elaeocarpus sylvestris)和黄桐(Endospermum chinense)为研究对象, 测定了1-5级细根的形态、解剖结构和组织碳(C)、氮(N)含量, 旨在探讨这些根系特征之间的联系。研究表明: 4个树种的细根形态差异较大, 但在树种水平上直径、根长和组织密度均随着根序的升高而增加, 比根长则随着根序的升高而降低; 低级根(前2级根或前3级根)具有皮层组织, 是典型的吸收根, 而高级根皮层组织消失, 是典型的运输和储藏根; 影响直径大小最重要的因子是皮层厚度, 它可以解释细根直径变异的97%, 而维管束直径仅能解释细根直径变异的70%; 根组织N和C浓度受维管束-根直径比(维根比)的影响, 随着维根比增加, 组织N浓度显著降低, 组织C浓度显著升高。4个树种细根的C/N比的变异受组织N浓度的影响程度为76%, 而受C浓度的影响程度不足10%。上述结果表明, 细根的形态特征、解剖结构和组织化学含量之间存在着紧密联系, 这为我们理解根系结构与功能变异提供了重要依据。     

Mixing and phase hold-ups variations due to gas production in anaerobic fluidized-bed digesters: influence on reactor performance

The influence of mixing and phase hold-ups on gas-producing fluidized-bed reactors was investigated and compared with an ideal flow reactor performance (CSTR). The liquid flow in the anaerobic fluidized bed reactor could be described by the classical axially dispersed plug flow model according to measurements of residence time distribution. Gas effervescence in the fluidized bed was responsible for bed contraction and for important gas hold-up, which reduced the contact time between the liquid and the bioparticles. These results were used to support the modeling of large-scale fluidized-bed reactors. The biological kinetics were determined on a 180-L reactor treating wine distillery wastewater where the overall total organic carbon uptake velocity could be described by a Monod model. The outlet concentration and the concentration profile in the reactor appeared to be greatly influenced by hydrodynamic limitations. The biogas effervescence modifies the mixing characteristics and the phase hold-ups. Bed contraction and gas hold-up data are reported and correlated with liquid and gas velocities. It is shown that the reactor performance can be affected by 10% to 15%, depending on the mode of operation and recycle ratio used. At high organic loading rates, reactor performance is particularly sensitive to gas effervescence effects. Copyright 1998 John Wiley & Sons, Inc.     

Root architecture in cultivated and wild lettuce (Lactuca spp.)

Root architecture is described for intact root systems of cultivated (Lactuca sativa L.) and wild (L. serriola L.) lettuce, grown for 5 to 6 weeks in greenhouse pot and cylinder experiments in coarse-textured soil. L. sativa cv. Salinas and a sclinas line of L. serriola attained the same biomass at 4 to 6 weeks after planting. Root biomass allocation was also similar, but root architecture differed. In the top zone along the tap root (0 to 5 cm), Salinas tended to produce more laterals, a greater total root length, and more external links (segments that originate at a branch point and end in a meristem) than wild lettuce. In the 5 to 55cm zone of the tap root, these measures were greater in the wild species. These patterns of root structure were generally corroborated by a second cylinder study with a different pair of L. sativa and L. serriola. Regressions of root structural traits were made against total root dry weight as a means to compare root architecture independently of the size of the root system. Regressions demonstrated that production of root segments differed between the two species; for example the slope for the regression of summed external link length in the top 0 to 5 cm with total root dry weight was significantly higher for Salinas, indicating that the rate of construction in the top 0 to 5cm was greater for cultivated than for wild lettuce. Yet, from 5 cm depth to the tap root tip, the rate of construction of external link length was greater in L. serriola. For many of these types of regression, r² and mean slope ± SE suggested that more variation occurred in cultivated than wild lettuce, yet genetic heterogeneity was probably low within the studied taxa. Inadvertent selection may have occurred in the breeding of cultivated lettuce varieties for increased root growth in the surface zone where water and fertilizer are applied, and for greater plasticity in construction of root segments, which might maximize the efficiency of exploitation of soil moisture and nutrients.     

Plant functional traits and soil microbial biomass in different vegetation zones on the Loess Plateau

Plant functional traits built the relationships between plant diversity, species composition, and physiology along with the environmental changes, thus influencing soil microbial community. As the sensitivity indicators, soil microbial biomass and plant functional traits responses soil micro-organism and plant characteristics in direct way. Ten plant functional traits of 149 species and soil microbial biomass (carbon, nitrogen, and phosphorus) were analyzed across the different vegetation types (forest, forest-steppe, and steppe) that are divided by environmental gradient (temperature and precipitation), aimed to find the correlations among them. Our results confirmed the greatest values of plant functional traits (except the leaf density and the fine root density) that were distributed in the steppe zone, mainly due to the different mean annual temperature and mean annual precipitation conditions. For different plant growth forms, the plant functional traits were significant differences among the vegetation zones. The advantages of higher rate nutrient cycling, plentiful biomass supplements, and favorite habit conditions lead to the forest-steppe zone with the highest C_mic and N_mic concentrations. The canonical correlation analysis indicated that leaf nitrogen, root nitrogen, and fine root densities were correlated with root exudate and tissue which affected the concentrations of soil organic carbon (SOC) and total nitrogen (N), consequently impacting soil microbial biomass carbon (C_mic) and soil microbial biomass nitrogen (N_mic). Soil is the medium that connects micro-organism and plant root system that influenced leaf nitrogen, root nitrogen, and fine root density of plant functional traits, the concentrations of SOC and total N that plant feedback are consequently influencing C_mic and N_mic.     

Controlled biomass formation and kinetics of toluene degradation in a bioscrubber and in a reactor with a periodically moved trickle-bed

The kinetics of degradation of toluene from a model waste gas and of biomass formation were examined in a bioscrubber operated under different nutrient limitations with a mixed culture. The applicability of the kinetics of continuous cultivation of the mixed culture was examined for a special trickle-bed reactor with a periodically moved filter bed. The efficiency of toluene elimination of the bioscrubber was 50 to 57% and depended on the toluene mass transfer as evident from a constant productivity of 0.026 g dry cell weight/L . h over the dilution rate. Under potassium limitation the biomass productivity was reduced by 60% to 0.011 g dry cell weight/L . h at a dilution rate of 0.013/h. Conversely, at low dilution rates the specific toluene degradation rates increased. Excess biomass in a trickle-bed reactor causes reduction of interfacial area and mass transfer, and increase in pressure drop. To avoid these disadvantages, the trickle-bed was moved periodically and biomass was removed with outflowing medium. The concentration of steady state biomass fixed on polyamide beads decreased hyperbolically with the dilution rate. Also, the efficiency of toluene degradation decreased from 72 to 56% with increasing dilution rate while the productivity increased. Potassium limitation generally caused a reduction in biomass, productivity, and yield while the specific degradation increased with dilution rate. This allowed the application of the principles of the chemostat to the trickle-bed reactor described here, for toluene degradation from waste gases. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 686-692, 1997.     

Prevention of clogging in a biological trickle-bed reactor removing toluene from contaminated air

Removal of organic compounds like toluene from waste gases with a trickle-bed reactor can result in clogging of the reactor due to the formation of an excessive amount of biomass. We therefore limited the amount of nutrients available for growth, to prevent clogging of the reactor. As a consequence of this nutrient limitation a lower removal rate was observed. However, when a fungal culture was used to inoculate the reactor, the toluene removal rate under nutrient limiting conditions was higher. Over a period of 375 days, an average removal rate of 27 g C/(m(3) h) was obtained with the reactor inoculated with the fungal culture. From the carbon balance over the reactor and the nitrogen availability it was concluded that, under these nutrient-limited conditions, large amounts of carbohydrates are probably formed. We also studied the application of a NaOH wash to remove excess biomass, as a method to prevent clogging. Under these conditions an average toluene removal rate of 35 g C/(m(3) h) was obtained. After about 50 days there was no net increase in the biomass content of the reactor. The amount of biomass which was formed in the reactor equaled the amount removed by the NaOH wash. (c) 1996 John Wiley & Sons, Inc.