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Applied Microbiology and Biotechnology (v.54, #1)


Molecular studies on isopenicillin N synthases by T. S. Sim; P. Loke (pp. 1-8).
The isopenicillin N synthases isolated thus far are related to oxidases from other microorganisms and plants. These enzymes maintain a non-heme monoferrous-dependent catalytic centre comprising a HisXAsp(53–57)XHis motif and a crucial substrate-binding pocket with an ArgXSer motif for their functionality. The elucidation of these motifs was dependent on information collated from studies on structural chemistry, structural biology, site-directed engineered mutations and biochemical experiments. It is envisaged that these enzymes can potentially be improved through molecular breeding and protein engineering.

Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures by O. Strauch; R.-U. Ehlers (pp. 9-13).
The entomopathogenic nematode–bacterium complex Heterorhabditis megidisPhotorhabdus luminescens was cultured in 10-l internal loop bioreactors with marine impellers at aeration rates of 0.3 vvm and 0.7 vvm. Process parameters like impeller velocity and oxygen saturation were controlled at equal set points. The bacterial density was assessed at 24 h. Nematode dauer juveniles (DJ) were then inoculated and the development to adults after 8 days and final DJ yields after 16 days were recorded. The bacterial population density and the nematode inoculum development was variable and was not influenced by the aeration rate. A significant effect on the yield was recorded at the highest aeration rate. This result was confirmed by a direct comparison in two 5-l internal loop glass bioreactors at 0.3 vvm and 1.0 vvm, which were inoculated with nematode and bacterium pre-cultures from the same flask culture. Possible reasons for the positive correlation between aeration rate and DJ yield are discussed.

Hydrodynamic aspects and Arthrospira growth in two outdoor tubular undulating row photobioreactors by P. Carlozzi (pp. 14-22).
Two tubular undulating row photobioreactors (TURPs) with a very high illuminated surface/volume ratio (400 m−1) were designed and constructed for the growth of photosynthetic micro-organisms. Experiments were conducted under outdoor conditions; and Arthrospira recycling was performed with airlifts (one for each row). The rows in each reactor faced east-west and consisted of a flexible polyvinyl chloride pipe (22 m long, 0.01 m bore) arranged in a sinusoidal shape. We studied the hydraulic performance of the sine-shaped photobioreactor rows during culture recycling in the TURPs at a very high Reynolds number (4200), when Arthrospira showed Newtonian fluid behavior. The sinusoidal pipe arrangement imposed a sine waveform on the culture, which led to better light utilization. During summer, a volumetric productivity of 2.2 g l−1 day−1 was reached in the TURP-5r (5 rows m−2), whereas an area productivity of 35 g m−2 day−1 was obtained in the TURP-10r (10 rows m−2). This was due to more light being available in the TURP-5r, because its rows were more spaced out and the photic ratio (R f) was low (3.0). In the TURP-10r, the closer rows caused a dilution of the sunlight, but gave a better light distribution inside the Arthrospira culture and improved the light utilization. This was attributed to the high R f (6.0) of this reactor.

Batch and continuous cultivation of Anaerobiospirillum succiniciproducens for the production of succinic acid from whey by P. C. Lee; W. G. Lee; S. Kwon; S. Y. Lee; H. N. Chang (pp. 23-27).
Batch and continuous cultivation of Anaerobiospirillum succiniciproducens were systematically studied for the production of succinic acid from whey. Addition of 2.5 g l−1 yeast extract and 2.5 g l−1 polypeptone per 10 g l−1 whey was most effective for succinic acid production from both treated and nontreated whey. When 20 g l−1 nontreated whey and 7 g l−1 glucose were used as cosubstrates, the yield and productivity of succinic acid reached at the end of fermentation were 95% and 0.46 g (l h)−1, respectively. These values were higher than those obtained using nontreated whey alone [93% and 0.24 g (l h)−1 for 20 g l−1 whey]. Continuous fermentation of A. succiniciproducens at an optimal dilution rate resulted in the production of succinic acid with high productivity [1.35 g (l h)−1], high conversion yield (93%), and higher ratio of succinic acid to acetic acid (5.1:1) from nontreated whey.

Characterisation of the shrinkage of calcium alginate gel membrane with immobilised Lactobacillus rhamnosus by W. Zhang; J. H. Kim; C. M. M. Franco; A. P. J. Middelberg (pp. 28-32).
To quantify the shrinkage of calcium alginate gel membrane as a support matrix for immobilising cells during the fermentation of Lactobacillus rhamnosus, factors including time, pH, membrane thickness, and the concentrations of immobilised cells, lactic acid, glucose, and calcium chloride were examined by statistical experimental design. A Plackett-Burman design was used for the first screening experiment to identify the important factors which caused the divergent effects. Uniform Design, a powerful modelling design technique, was thus chosen to design the modelling experiments. Through a non-linear step-wise regression analysis, the predictive mathematical model of the shrinkage in membrane thickness was established and the significant main effects and two-factor interactions were identified. However, no significant model equations could be obtained for the shrinkage in area and volume of gel membranes. The methodology developed can be extrapolated to the quantitative characterisation of shrinkage in other immobilised gel matrices, which will be very useful in mathematical modelling, design, operation and scale-up of gel immobilised cell systems.

Growth of Streptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production by A. Fang; D. L. Pierson; S. K. Mishra; A. L. Demain (pp. 33-36).
Growth of Streptomyces hygroscopicus under conditions of simulated microgravity in a rotating-wall bioreactor resulted in a pellet form of growth, lowered dry cell weight, and inhibition of rapamycin production. With the addition of Teflon beads to the bioreactor, growth became much less pelleted, dry cell weight increased but rapamycin production was still markedly inhibited. Growth under simulated microgravity favored extracellular production of rapamycin, in contrast to a greater percentage of cell-bound rapamycin observed under normal gravity conditions.

In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa by Q. Qi; A. Steinbüchel; B. H. A. Rehm (pp. 37-43).
For the first time, the purification has been achieved of the type II polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa applying N-terminal His6-tag fusions and metal chelate affinity chromatography. In vivo His6-tagged PHA synthase activity was confirmed by functional expression of the corresponding genes in Escherichia coli, and PHA synthase activity could also be measured in vitro with the enzymes. The specific enzyme activity of PHA synthases PhaC1 and PhaC2 was 0.039 U mg−1 and 0.035 U mg−1 protein, respectively. Kinetic studies showed a lag phase for both PHA synthases using (R,S)-3-hydroxydecanoyl-CoA as substrate. Specific enzyme activity was increased to 0.055 U mg−1 when the phasin GA24 from Ralstonia eutropha was added to the assay. CoA inhibited PHA synthase activity, and a K i of 85 μM was determined. A two-enzyme system was established, employing commercially available acyl-CoA synthetase and PHA synthase, which allowed the in vitro de novo PHA granule formation and the in vitro synthesis of poly(3-hydroxydecanoate) exhibiting a weight average molar mass of 9.8 × 104 g mol−1, and which occurred independently of pre-existing PHA granules.

Gene cloning and overproduction of low-specificity d-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug by J. Q. Liu; M. Odani; T. Yasuoka; T. Dairi; N. Itoh; M. Kataoka; S. Shimizu; H. Yamada (pp. 44-51).
The dtaAX gene encoding a pyridoxal 5′-phosphate (pyridoxal-P)-dependent low-specificity d-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of d-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity d-threonine aldolase was shown to be an efficient biocatalyst for resolution of l-β-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.

Expression in Escherichia coli, purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32 by W.-W. Zhang; W.-H. Jiang; G.-P. Zhao; Y.-L. Yang; J.-S. Chiao (pp. 52-58).
The operon encoding aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask ) and E. coli X6118 (asd ), respectively. The A. mediterranei U32 aspartokinase and aspartate semialdehyde dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and aspartate semialdehyde dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has K m values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has K m values of 1.25 mM for dl-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by l-threonine, l-lysine, and l-methionine, but not by l-isoleucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that aspartokinase was subject to allosteric control by l-threonine. Repression of both aspartokinase and aspartate semialdehyde dehydrogenase gene transcription in A. mediterranei U32 by l-lysine, l-methionine, l-threonine, and l-isoleucine were found. The network of regulation of aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae by K. Koibuchi; H. Nagasaki; A. Yuasa; J. Kataoka; K. Kitamoto (pp. 59-68).
 A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of l-glutamine but also of d-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137–143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5′-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae.

Overexpression of the lys1 gene in Penicillium chrysogenum: homocitrate synthase levels, α-aminoadipic acid pool and penicillin production by O. Bañuelos; J. Casqueiro; S. Gutiérrez; J. F. Martín (pp. 69-77).
 Homocitrate synthase activity (encoded by the lys1 gene) catalyzes the first step of the lysine and penicillin pathway and is highly sensitive to feedback regulation by l-lysine. The transcript levels of the lys1 gene and the homocitrate synthase activity are high during the growth phase and decrease during the antibiotic production phase, except in the high penicillin producer strain AS-P-99 which maintained high levels of homocitrate synthase activity in cultures at 96 h and 120 h. The lys1 gene was overexpressed in Penicillium chrysogenum using additional copies of lys1 with its own promoter or under the control of the pcbC promoter in either autonomously replicating or integrative vectors. Transformants containing 3 to 32 additional copies of the lys1 gene were selected. Some of these transformants, particularly Ti-c4 (integrative) and Tar-l9 (with autonomously replicating plasmids) showed very high levels of lys1 transcript and, in the case of Tar-l9, high levels of homocitrate synthase activity in cultures of 120 h. However, these transformants did not show increased α-aminoadipate or lysine pools. A mutant P. chrysogenum LG disrupted in the lys2 gene (therefore lacking the lysine branch of the pathway) showed increased α-aminoadipate levels and produced higher levels of penicillin than non-disrupted control strains. Overexpression of the lys1 gene in the LG mutant resulted in high homocitrate synthase levels but no additional increase of the α-aminoadipate pool or penicillin production levels. These results suggest that after amplification of the homocitrate synthase levels there are other limiting steps in the common stem of the lysine and penicillin pathways.

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase by C.-F. Wu; H. J. Cha; G. Rao; J. J. Valdes; W. E. Bentley (pp. 78-83).
 Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence.

Molecular design of novel metal-binding oligomeric human metallothioneins by S.-H. Hong; M. Gohya; H. Ono; H. Murakami; M. Yamashita; N. Hirayama; Y. Murooka (pp. 84-89).
 Genes for dimeric and tetrameric human metallothionein (hMT) were designed and successfully overexpressed in Escherichia coli to generate functional oligomeric hMTs. An hMT synthesized with prokaryotic codons, a linker encoding a gly-gly-gly tripeptide, and Met-deficient hMT-II was ligated to create a dimeric hMT, from which a tetrameric hMT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. The oligomeric proteins formed inclusion bodies which were dissolved with dithiothreitol, and the purified apo-metallothioneins were reconstituted with Cd or Zn ions in a reducing condition. The oligomeric hMT proteins incubated with Cd ions showed a typical Cd-thiolate absorbance peak at 245–255 nm. The dimeric and tetrameric hMT proteins exhibited both Cd and Zn binding activities that were respectively two and four times higher than those of the hMT-II monomer protein. These novel oligomeric hMTs may be useful in bioremediation for heavy metals.

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface by K. Ye; S. Shibasaki; M. Ueda; T. Murai; N. Kamasawa; M. Osumi; K. Shimizu; A. Tanaka (pp. 90-96).
An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence.

Construction of a flocculent brewer's yeast strain secreting Aspergillus nigerβ-galactosidase by L. Domingues; M.-L. Onnela; J. A. Teixeira; N. Lima; M. Penttilä (pp. 97-103).
One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being the flocculating yeast production system. Also, lactose is available in large amounts as a waste product from cheese production processes. The construction of flocculent and non-flocculent brewer's yeast strains secreting β-galactosidase and growing on lactose is presented. A plasmid was constructed coding for an extracellular β-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper. This selective marker allows for the transformation of wild-type yeasts. This work represents an important step towards the study of heterologous protein secretion by flocculent cells.

Polymerization of guaiacol by lignin-degrading manganese peroxidase from Bjerkandera adusta in aqueous organic solvents by K. Iwahara; Y. Honda; T. Watanabe; M. Kuwahara (pp. 104-111).
 Lignin-degrading manganese (II) peroxidase (MnP) purified from the culture of a wood-rotting basidiomycete, Bjerkandera adusta, was used in the polymerization of guaiacol. MnP was found to catalyze polymerization of guaiacol in 50% aqueous acetone, dimethyl formamide, methanol, ethanol, dioxane, acetonitrile, ethylene glycol and methylcellosolve. Maximum yield of polyguaiacol was achieved in 50% aqueous acetone. The weight average molecular weight (M w) of the polymer was estimated to be 30 300 by gel permeation chromatography. However, matrix-assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF-MS) analysis gave a more reliable M w of 1690. IR, 13C-NMR, MALDI-TOF-MS and pyrolysis GC-MS analyses showed the presence of C–C and C–O linkages and quinone structure in polyguaiacol. It was also indicated that polyguaiacol has a methoxy-phenyl group as the terminal moiety. This suggests that polyguaiacol is a branched polymer in which guaiacol units are cross-linked at the phenolic group. Thermal gravimetric and differential scanning calorimetric analyses were also carried out. MnP also catalyzed the polymerization of o-cresol, 2,6-dimethoxyphenol and other phenolic compounds and aromatic amines. M w of these polymers ranged from around 1000 to 1500.

Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. UTEX LB1999 by M. Takagi; K. Watanabe; K. Yamaberi; T. Yoshida (pp. 112-117).
Limited feeding of nitrate during culture of Nannochloris sp. UTEX LB1999 for intracellular lipid and triglyceride accumulation was investigated with the aim of obtaining cells superior for liquefaction into a fuel oil. The intracellular lipid contents and the percentage of triglycerides in the lipids of cells grown in a nitrogen-limited medium (0.9 mM KNO3) were 1.3 times as high as those grown in a modified NORO medium containing 2.0–9.9 mM KNO3. However, the cell concentration was too low for the practical production of fuel oil by high-pressure liquefaction of the cell mass. A single feeding of 0.9 mM nitrate after nitrate depletion during cultivation in a nitrate-limited medium increased the cell concentration to twice that obtained without such feeding, and the lipid content was maintained at a high level. The timing of nitrate feeding, i.e., whether it was given during the log phase (before nitrate depletion), the constant growth phase (just after the depletion), or the stationary phase (after the depletion), had negligible effect on the intracellular lipid content and percentage of triglycerides in the lipids. When 0.9 mM nitrate was intermittently fed ten times during the log phase in addition to the initial nitrate feed (0.9 mM), the cell concentration reached almost the same (2.16 g/l) and the intracellular lipid content and the percentage of triglycerides in the lipids increased from 31.0 to 50.9% and 26.0 to 47.6%, respectively, compared with those of cells cultured in a modified NORO medium containing 9.9 mM KNO3 without additional nitrate feeding.

The effect of heat-shocking on batch fermentation by Clostridium beijerinckii NRRL B592 by J. R. Gapes; H. Swoboda; A. Haslinger; D. Nimcevic (pp. 118-120).
 In spite of the large-scale industrial use of the acetone-butanol fermentation process earlier this century (until 1983 in South Africa), very little has been published on the inoculum preparation techniques required for successful fermentation using these bacteria. In particular, heat-shocking has often been referred to as “useful” but no quantitative data are available. Data presented in this paper demonstrate and quantify the effect of heat-shocking on batch fermentation yields using one organism capable of this fermentation.

Biodegradability of volatile hydrocarbons of gasoline by F. Solano-Serena; R. Marchal; T. Huet; J.-M. Lebeault; J.-P. Vandecasteele (pp. 121-125).
The biodegradability under aerobic conditions of volatile hydrocarbons (4–6 carbons) contained in gasoline and consisting of n-alkanes, iso-alkanes, cycloalkanes and alkenes, was investigated. Activated sludge was used as the reference microflora. The biodegradation test involved the degradation of the volatile fraction of gasoline in closed flasks under optimal conditions. The kinetics of biodegradation was monitored by CO2 production. Final degradation was determined by gas chromatographic analysis of all measurable hydrocarbons (12 compounds) in the mixture after sampling the headspace of the flasks. The degradation of individual hydrocarbons was also studied with the same methodology. When incubated individually, all hydrocarbons used as carbon sources, except 2,2-dimethylbutane and 2,3-dimethylbutane, were completely consumed in 30 days or less with different velocities and initial lag periods. When incubated together as constituents of the light gasoline fraction, all hydrocarbons were metabolised, often with higher velocities than for individual compounds. Cometabolism was involved in the degradation of dimethyl isoalkanes.

Competition between n-alkane-assimilating yeasts and bacteria during colonization of sandy soil microcosms by C. Schmitz; I. Goebel; S. Wagner; A. Vomberg; U. Klinner (pp. 126-132).
An n-alkane-assimilating strain of Candida tropicalis was selected in sandy soil inoculated with microorganisms from contaminated sites. Competition experiments with n-alkane utilizers from different strain collections confirmed that yeasts overgrow bacteria in sandy soil. Acidification of the soil is one of the colonization factors useful for the yeasts. It can be counteracted by addition of bentonite, a clay mineral with high ion exchange capacity, but not, however, by kaolin. Strains of different yeast species showed different levels of competitiveness. Strains of Arxula adeninivorans, Candida maltosa, and Yarrowia lipolytica overgrew strains of C. tropicalis, C. shehatae or Pichia stipitis. Two strains of C. maltosa and Y. lipolytica coexisted during several serial transfers under microcosm conditions.

Toluene degradation in the recycle liquid of biotrickling filters for air pollution control by H. H. J. Cox; T. T. Nguyen; M. A. Deshusses (pp. 133-137).
Pollutant degradation in biotrickling filters for waste air treatment is generally thought to occur only in the biofilm. In two experiments with toluene degrading biotrickling filters, we show that suspended microorganisms in the recycle liquid may substantially contribute to the overall pollutant removal. Two days after reactor start up, the overall toluene elimination capacity reached a maximum of 125 g m−3 h−1, which was twice that found during prolonged operation. High biodegradation activity in the recycle liquid fully accounted for this short-term peak of pollutant elimination. During steady-state operation, the toluene degradation in the recycle liquid was 21% of the overall elimination capacity, although the amount of suspended biomass was only 1% of the amount of immobilized biomass. The results suggest that biotrickling filter performance may be improved by selecting operating conditions allowing for the development of an actively growing suspended culture.
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