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Applied Microbiology and Biotechnology (v.47, #5)
Biosynthesis and chemical reactions of poly(amino acid)s from microorganisms by M. Kunioka (pp. 469-475).
The biosynthesis and chemical reactions of poly(amino acid)s produced by microorganisms are reviewed. A large amount of γ-poly(glutamic acid) (PGA) has been produced by Bacillus strains. ε-Polylysine (PL) has been produced by Streptomyces albulus. As a modification of PGA and PL, pH-sensitive hydrogels have been prepared by means of γ irradiation or the addition of a crosslinking agent to an aqueous solution of PGA and PL.
Continuous sucrose hydrolysis by yeast cells immobilized to wool by A. Krastanov (pp. 476-481).
A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 103kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.
Cell-dislodging methods under serum-free conditions by H. J. Cruz; E. M. Dias; J. L. Moreira; M. J. T. Carrondo (pp. 482-488).
In this work, a BHK21 clone producing a fusion protein consisting of a recombinant human IgG molecule with a cytokine tail, growing in a protein-free medium, was used to test several alternatives to avoid the use of serum for trypsin inactivation, currently used in cell dislodging. These included (1) trypsin inactivated with soybean trypsin inhibitor (STI); (2) cell dissociation solution instead of trypsin; (3) dispase instead of trypsin; (4) trypsin inactivated with fetal calf serum (positive control); (5) non-inactivated trypsin (negative control). Use of a centrifugation step was also tested for each alternative. Results indicate that the best method regarding cell growth, viability and adherent fraction is to use trypsin inactivated with STI followed by a centrifugation step. For all methods tested, the utilization of a centrifugation step always led to improved results. The optimal proportion for total trypsin inactivation is 1:1 trypsin (0.2% w/v) to STI (1 mg ml−1), equivalent to 2 mg trypsin to 1 mg STI. No toxic effect was observed for STI at the concentrations used. Long-term subculturing with this new, alternative dislodging method did not affect cell growth, viability and productivity.
Pyrazine production by Bacillus subtilis in solid-state fermentation on soybeans by I. Besson; C. Creuly; J. B. Gros; C. Larroche (pp. 489-495).
2,5-Dimethylpyrazine (2,5-DMP) and tetramethylpyrazine (TTMP) were produced using Bacillus subtilis IFO 3013 grown on soybeans. Solid-state cultivations were carried out either in 100-ml bottles or in a fixed-bed column reactor, both systems being at 27 °C. Optimization studies showed that the best way to produce the two above aroma compounds involved two separate processes. 2,5-DMP was obtained using soybeans enriched with 75 g threonine/kg initial dry weight (i.d.w.), giving 0.85 g metabolite/kg i.d.w. after 6 days. TTMP production involved addition of 90 g/kg i.d.w. acetoin to soybeans, and 2.5 g/kg i.d.w. was recovered after 14 days. These results demonstrated the suitability of solid-state cultivation for production of high-added-value compounds.
Sophorose lipid fermentation with differentiated substrate supply for growth and production phases by A.-M. Davila; R. Marchal; J.-P. Vandecasteele (pp. 496-501).
Sophorose lipid production by Candida bombicola is a two-step process where sophorose lipids are mainly produced after a first stage of growth, ending because of nitrogen limitation. The influence of the following parameters was individually studied for both the stages of growth and of product formation with respect to final sophorose lipid production performance: pH, temperature and carbon source. Glucose and rapeseed ethyl esters were supplied individually or as a dual carbon source. The lipidic substrate was added by continuous feeding. It was found that supplying both carbon sources during the production step was crucial for obtaining a high production performance ranging from 250 g l−1 to 300 g l−1 or more. Controlling the feeding of rapeseed ethyl esters to avoid inhibition by fatty acids was essential for a successful scale-up of the fermentation on the industrial scale. The conditions of substrate feeding markedly affected the composition of the mixture of sophorose lipids produced, namely the extent of acetylation of the sophorose moieties and distribution of the acidic and lactonic forms. The results suggest that the physiological role of sophorose lipid production is related to the regulation of energy metabolism.
Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense by H. M. Kalisz; J. Hendle; R. D. Schmid (pp. 502-507).
Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH4)2SO4, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the V max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V max and a 2-fold decrease in K m were observed.
Pyranose 2-oxidase from Phanerochaete chrysosporium– further biochemical characterisation by M. J. Artolozaga; E. Kubátová; J. Volc; H. M. Kalisz (pp. 508-514).
Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (k cat) of 111.0 s−1 and an affinity constant (K m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k cat/K m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k cat decreased to 60.9 s−1 and the K m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.
Production, purification, and characterization of a highly enantioselective (S )-N-acetyl-1-phenylethylamine amidohydrolase from Rhodococcus equi Ac6 by A. Brunella; M. Graf; M. Kittelmann; K. Laumen; O. Ghisalba (pp. 515-520).
Rhodococcus equi Ac6 was found to express an inducible (S )-specific N-acetyl-1-phenylethylamine amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed around pH 6.5. Purification of the enzyme to a single band in a Coomassie-blue-stained sodium dodecyl sulfate/polyacrylamide gel (SDS-PAGE) was achieved by ammonium sulfate precipitation of R. equi Ac6 crude extract and column chromatographies on Fractogel TSK Butyl-650(S) and Superose 12HR. At pH 7.0 and 30 °C the amidohydrolase had a half-life of around 350 days; at 44 °C it was only 10 min. Except for Ni2+ and, to some extent, Zn2+ and Co2+, the enzyme was neither strongly influenced by metal cations nor by chelating agents, but was inhibited by 95% at 0.1 mM phenylmethylsulfonyl fluoride. The molecular mass of the native enzyme was estimated to be 94 kDa by gel filtration and 50 kDa by SDS-PAGE, suggesting a dimeric structure. Specificity experiments revealed a spectrum of related N-acetylated compounds being hydrolyzed with variable enantiomeric selectivities.
Purification of α-amylase by specific elution from anti-peptide antibodies by S. Katoh; M. Terashima; K. Miyaoku (pp. 521-524).
Chimeric α-amylase, produced by recombinant yeast cells, was purified by immunoaffinity chromatography by use of an anti-peptide antibody and an eluent containing an antigen peptide. Chimeric α-amylase was adsorbed by the antibody against the peptide corresponding to the C-terminal region of target α-amylase, and specifically eluted by the eluent containing the antigen peptide used for immunization. A low concentration of the peptide could competitively elute adsorbed α-amylase, and the rate-limiting step of the elution was mass transfer of desorbed α-amylase. With this specific method, target proteins can be effectively eluted, and highly purified under mild conditions, from the antibody ligand showing a high-affinity for the adsorption step.
Growth and nodulation competitiveness of Sinorhizobium meliloti L1 (RecA−) is less than that of its isogenic strain L33 (RecA+) but comparable to that of two S. meliloti wild-type isolates by S. Niemann; A. Pühler; W. Selbitschka (pp. 525-529).
Gnotobiotic systems were used to assess the competitive abilities of bioluminescent Sinorhizobium meliloti strains L1 (RecA−) and L33 (RecA+) for growth and host plant nodulation in the presence of a reconstructed S. meliloti population. Three wild-type strains belonging to infective subgroups of a natural S. meliloti population were chosen as competitors in microcosm studies. Whereas the RecA+ strain L33 dominated the reconstructed population with respect to growth and alfalfa nodulation, the competitiveness of the RecA− strain L1 was reduced compared to that of one of the field strains, but comparable to that of the other field isolates. This result indicates that strain L1, despite its recA mutation, has the potential to compete successfully with a resident S. meliloti population after environmental release.
Controlled secretion into the culture medium of a hybrid -glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5 -derived transposon by G. Miksch; E. Fiedler; P. Dobrowolski; E. Flaschel (pp. 530-536).
A Tn5-based transposon bearing the kil gene (killing protein), mediating controlled export of periplasmic proteins into the culture medium, was constructed (Tn5-KIL3). This transposon contained the kil gene of the ColE1 plasmid under the growth-phase-dependent promoter of the fic gene (filamentation induced by cAMP) of Escherichia coli, an interposon located upstream of kil, a kanamycin/neomycin-resistance gene, a multiple cloning site and the mob site. The transposition of Tn5-KIL3 to Acetobacter methanolicus showed a moderate transposition frequency (10−5–10−6). By insertion of a Bacillus hybrid β-glucanase (bgl ) as a model protein into the transposon (Tn5-LF3) it was shown that the secretion function as well as the gene of the target protein had been transferred to and stably integrated into the chromosome of A. methanolicus, and that the transposition of Tn5-LF3 was non-specific. β-Glucanase was highly overexpressed and secreted into the medium during stationary phase. Total and extracellular production of β-glucanase varied depending on the integration site of the transposon. The viability of the bacterial cells was not affected, and cell lysis did not occur.
Protein A as a fusion partner for the expression of heterologous proteins in Lactobacillus by C. Rush; L. Hafner; P. Timms (pp. 537-542).
An expression system based on the Staphylococcus aureus protein A gene (spa) was developed to allow the production and export of proteins in Lactobacillus. Plasmid shuttle vectors were constructed that carried the eZZ gene, a synthetic gene based on the Protein A gene (spa) but lacking the carboxy-terminal membrane-anchoring region. A gene fusion was created between the eZZ gene and the VD4 region of a chlamydial major outer-membrane protein gene. Expression studies demonstrated the recognition of the spa regulatory signals by several Lactobacillus, with the recombinant protein being expressed (from 0.1 μg of EZZVD4 fusion protein per ml in L. plantarum up to 10 μg of EZZ protein per ml in L. fermentum) and exported (levels up to 20% in L. fermentum) in several Lactobacillus strains.
Plasmids for efficient single-copy gene cloning into gdh2 and trpC of Bacillus megaterium DSM319 and QM B1551 by D. Schmiedel; P. S. Vary; L. Jablonski; W. Hillen (pp. 543-546).
We constructed integrative plasmids to place xylA-lacZ indicator gene fusions into two different loci of the Bacillus megaterium chromosome, gdh2 and trpC, in lac mutants of strains DSM 319 and QM B1551, which differ markedly. Single-crossover integration was achieved in all cases while double crossovers occurred only in gdh2 of DSM 319 and QM B1551 and in trpC of QM B1551. Neither of the loci affected regulation of the xylA-lacZ fusions. These results confirm the suitability of the two gene loci for single-copy cloning.
Influence of aeration and carbon source on production of microcin B17 by Escherichia coli ZK650 by A. Fang; A. L. Demain (pp. 547-553).
Previous studies [Connell et al. (1987) Mol Microbiol 1: 195–201] have shown that expression of the microcin B17 (MccB17) promoter is inversely related to the growth rate of the culture, when slower growth was brought about by limitation of sources of carbon, nitrogen or phosphorus. When we used oxygen limitation to decrease growth in a glucose-based chemically defined medium, we found specific MccB17 production to be positively related to growth rate and extent. On the other hand, when we examined various nutritional variations of media, specific production of MccB17 showed a negative relationship to growth rate and extent, as would be predicted by the findings of Connell et al. (1987). Glucose, glycerol and acetate were found to repress MccB17 production; succinate was not repressive. Succinate is an excellent carbon source for production of MccB17 since high levels can be used with no or little interference in product synthesis.
Metabolic response of Bacillus sphaericus 1593M for dual-substrate limitation in continuous and total-cell-retention cultures by S. Meenakshisundaram; G. Suresh; R. K. Fernando; K. Jenny; R. Sachidanandham; K. Jayaraman (pp. 554-559).
A chemically defined medium has been developed to support the growth and the production of mosquito larvicidal factor(s) (MLF) of Bacillus sphaericus 1593M. On the basis of the data of steady-state continuous cultures, it has been understood that acetate can serve as a sole carbon and energy source for B. sphaericus 1593M. Utilization of acetate by B. sphaer-icus 1593M and the production of MLF are further enhanced by the addition of glutamate at low concentrations, both in steady-state continuous as well as in total-cell-retention cultures (TCRC). A two-step TCRC procedure resulted in better biomass and MLF production by B. sphaericus 1593M. It was also found that glutamate can serve as a carbon source as well as a growth factor in the presence of acetate and hence is a partially substitutable carbon source.
3-Ketoglycoside-mediated metabolism of sucrose in E. coli as conferred by genes from Agrobacterium tumefaciens by P. L. Schuerman; J. S. Liu; H. Mou; A. M. Dandekar (pp. 560-565).
Escherichia coli strains that did not have the ability to use sucrose as a sole carbon source gained this ability after receiving a cloned fragment of DNA from Agrobacterium tumefaciens. No invertase was detected in the sucrose-metabolizing E. coli, but evidence for the activity of certain enzymes, known to be produced by biotype 1 strains of Agrobacterium, were found. Evidence was found for the presence of d-glucoside 3-dehydrogenase (G3DH) and α-3-ketoglucosidase. The activity of enzyme extracts on 3-ketosucrose also indicated that 3-ketoglucose reductase, or some enzyme that acts on 3-ketoglucose, was present in the Suc+ E. coli as well. The fragment was found to complement a G3DH mutant of A. tumefaciens and was also found to confer chemotaxis towards sucrose in E. coli.
Depolymerization of low-rank coal by extracellular fungal enzyme systems. III.In vitro depolymerization of coal humic acids by a crude preparation of manganese peroxidase from the white-rot fungus Nematoloma frowardii b19 by M. Hofrichter; W. Fritsche (pp. 566-571).
The in vitro depolymerization of humic acids derived from German lignite (low-rank coal, brown coal) was studied using a manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii b19. The H2O2 required was continuously generated by glucose oxidase. Mn peroxidase depolymerized high-molecular-mass humic acids by forming fulvic-acid-like compounds. The depolymerization process was accompanied by the decolorization of the dark-brown humic acid fraction soluble in alkaline solutions (decrease in absorbance at 450 nm) and by the yellowish coloring of the fraction of acid-soluble fulvic-acid-like compounds (increase in absorbance at 360 nm). The Mn peroxidase of N. frowardii b19 has been proved to be highly stable; even after an in vitro reaction time of 7 days in the presence of humic acids, less than 10% loss in total oxidizing activity was detectable.
Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems by G. Misra; S. G. Pavlostathis (pp. 572-577).
Batch experiments were conducted to evaluate the biodegradation rates of limonene, α-pinene, γ-terpinene, terpinolene and α-terpineol at 23 °C under aerobic conditions. Biodegradation was demonstrated by the depletion of monoterpene mass, CO2 production and a corresponding increase in biomass. Monoterpene degradation in liquid cultures devoid of soil followed Monod kinetics. The maximum specific growth rate (μmax) was 0.02 h−1 and 0.06 h−1 and the half-velocity constant (K s ) varied from 32 mg/l to 3 mg/l for the limonene and α-terpineol respectively. The recovery of monoterpenes by solvent extraction from autoclaved and azide-amended soil-slurry samples decreased over time and ranged from 69% to 73% for 120 h of incubation period. Although a significant fraction of monoterpene hydrocarbon could not be extracted, mineralization of these compounds in the soil-slurry systems took place, as shown by CO2 production. The soil-normalized degradation rates for the hydrocarbon monoterpenes ranged from 0.6 μg g−1 h−1 to 2.1 μg g−1 h−1. A kinetic model – which combined monoterpene biodegradation in the liquid phase and net desorption – was developed and applied to data obtained from soil-slurry assays.
Identification of Rhodococcuserythropolis isolates capable of degrading the fungicide carbendazim by M. A. Holtman; D. Y. Kobayashi (pp. 578-582).
Continuous enrichment cultures were used to identify bacterial isolates capable of degrading the fungicide carbendazim. The bacteria originated from sites that had been repeatedly treated with the structurally related parent fungicide, benomyl, over a period of several years. Six isolates were identified as carbendazim degraders on the basis of their ability to produce diffusion-clearing zones on a minimal salts solid medium spray-coated with a 0.1% solution of carbendazim, their ability to grow in a minimal salts broth supplemented with carbendazim as the sole carbon source, and their ability to reduce carbendazim levels in liquid cultures. All six isolates were identified as Rhodococcus erythropolis or a closely related species by analyses of nutritional utilization and whole-cell fatty acid methyl ester profiles. A chemically induced mutant of R. erythropolis isolate B2E was identified that was no longer capable of degrading carbendazim, as determined by negative results in all three assays. Further characterization of these strains provides an opportunity for their development in bioremediation of the compound.
Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization by M. Sojo; R. Bru; D. Lopez-Molina; F. Garcia-Carmona; J.-C. Argüelles (pp. 583-589).
Rhodococcus erythropolis cells growing in a cholesterol-free glycerol-containing mineral medium displayed very low levels of a cell-wall-bound cholesterol oxidase activity. Addition of cholesterol induced a marked increase in the synthesis of this enzyme, which reached a maximum within 6 days and was subsequently followed by the appearance of extracellular cholesterol oxidase in the culture broth. Significant levels of induction were only achieved when cholesterol emulsified with Tween 80. The presence of chloramphenicol at the time of induction completely prevented the emergence of both enzymatic forms, suggesting the requirement of de novo protein synthesis. Upon transfer of cholesterol-growing cultures to fresh medium lacking cholesterol, the extracellular cholesterol oxidase was quickly erased, while the activity of the particulate enzyme decreased sharply. The electrophoretic pattern on native Western blotting as well as on sodium dodecyl sulphate/polyacrylamide gels, together with kinetic data, strongly support the idea that the particulate and extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa.
Production of cellulase by a wild strain of Chaetomium globosumusing delignified oil palm empty-fruit-bunch fibre as substrate by M. S. Umikalsom; A. B. Ariff; Z. H. Shamsuddin; C. C. Tong; M. A. Hassan; M. I. A. Karim (pp. 590-595).
Studies on the feasibility of using delignified oil palm empty-fruit-bunch (OPEFB) fibres as a substrate for cellulase production by Chaetomium globosum strain 414 were carried out in shake-flask cultures containing different types and concentrations of nitrogen source. Peptone, as nitrogen source, gave maximum production of all the three main components of the cellulase complex (endoglucanase or carboxymethylcellulase, cellobiohydrolase or filter-paper-hydrolysing enzyme and β-glucosidase), followed by yeast extract, urea, KNO3 and (NH4)2SO4. The maximum specific growth rate (μmax) of C. globosum strain 414 grown in medium containing OPEFB and peptone was 0.038 h−1. In all the fermentations, the fungus was able to produce all the three cellulases with significant amounts of β-glucosidase, except when using (NH4)2SO4 as nitrogen source, where β-glucosidase was not produced. With 6 g/l peptone and 10 g/l delignified OPEFB fibres, the fungus produced maximum concentrations of FPase, carboxymethylcellulase and β-glucosidase: 1.4, 30.8 and 9.8 U/ml, giving productivities of 10, 214 and 24 U l−1h−1, respectively. The cellulase mixture, partially purified by ammonium sulphate precipitation, was able to hydrolyse delignified OPEFB fibres, converting about 68 % of the cellulosics to reducing sugars after 5 days.
Oxidation of polycyclic aromatic heterocycles by Pseudomonas fluorescens TTC1 by D. Bianchi; A. Bosetti; D. Cidaria; A. Bernardi; I. Gagliardi; P. D'Amico (pp. 596-599).
The mutant strain Pseudomonas fluorescens TTC1 (NCIMB 40605), derived from the naphthalene-degrading P. fluorescens N3 (NCIMB 40530), was used for the biotransformation of polycyclic aromatic heterocycles such as dibenzothiophene, dibenzofuran, thianthrene xanthen and acridine. The cis-1,2- and cis-3,4-dihydrodiols produced were isolated and identified from the culture filtrate. Both the regioselectivity and the productivity of the transformations, catalysed by the naphthalene dioxygenase enzymatic system, were dramatically influenced by the presence of the heteroatom. The high substrate tolerance displayed by the enzyme might be useful in the biotransformation of other related compounds.
Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum by H. Dominguez; M. Cocaign-Bousquet; N. D. Lindley (pp. 600-603).
Growth of Corynebacterium glutamicum on mixtures of glucose and fructose leads to simultaneous consumption of both sugars in which the uptake of each sugar is directly related to the expression of the corresponding sugar uptake mechanism. The overall rate of sugar uptake was higher on sugar mixtures than on either glucose or fructose alone and was similar to that observed during sucrose metabolism. The results suggest that sugar uptake limits metabolic rates though, in the case of fructose, overflow metabolism of both lactate and dihydroxyacetone was observed. Such products could reflect a higher flux through glycolysis rather than the pentose pathway during catabolism of fructose.
Bioluminescence induction response and survival of the bioreporter bacterium Pseudomonas fluorescens HK44 in nutrient-deprived conditions by U. Matrubutham; J. E. Thonnard; G. S. Sayler (pp. 604-609).
Pseudomonas fluorescens HK44 is a bioluminescent bioreporter synthesizing light in the presence of naphthalene or salicylate. Upon immobilization, HK44 is useful as an in situ or on-line biosensor of bioavailable naphthalene and salicylate in waste streams or contaminated fields. The bioreporting efficacy of alginate/SrCl2-immobilized HK44 was investigated in simulated groundwater with different pH regimes. When induced with complex (salicylate plus auxiliary energy supplements) and simple (salicylate as the sole energy supplement) inducer solutions, the specific light response was steadier at pH 6 than at pH 7 in a 35-day study. There was no bioluminescence response from cells incubated in groundwater samples with pH below 6. The rate of the luminescence reaction was stable at pH 6 irrespective of the type of inducer solution, indicating the robust physiological status of the bioreporter bacteria. In addition, the quantity of light synthesized was at least one order of magnitude higher with complex inducer solution than with simple inducer solution. The numbers of viable and cultivable cells remained constant in groundwater at pH 6 and 7 (approx. 107 g−1 beads). The numbers decreased by four orders of magnitude (107 to 103) to zero in groundwaters with pH below 6. This study suggested that HK44 is useful for long-term biosensor applications in moderately acidic to neutral groundwater conditions.
Multi-substrate growth kinetics of Pseudomonas putida for phenol removal by Ş. Şeker; H. Beyenal; B. Salih; A. Tanyolaç (pp. 610-614).
The biodegradation of phenol by a pure culture of Pseudomonas putida was investigated in a continuously fed stirred-tank reactor, under aerobic conditions. The dilution rate was varied between 0.0174 h−1 and 0.278 h−1, covering a wide range of dissolved oxygen and the inhibition region of phenol. Through non-linear analysis of the data, a dual-substrate growth kinetics, Haldane kinetics for phenol and Monod kinetics for oxygen, was derived with high correlation coefficients. Respective biokinetic parameters were evaluated as μm = 0.569 h−1, K p = 18.539 mg/l, K i = 99.374 mg/l, K o = 0.048 mg/l, Y x/p = 0.521 g microorganism/g phenol and Y x/o = 0.338 g microorganism/g oxygen, being in good agreement with other studies in the literature. Maintenance factors for both phenol and oxygen were calculated for the first time for P. putida while the saturation coefficient for oxygen, K o, was genuinely evaluated from the constructed model, not imported or adapted from other studies as reported in the literature. All pertinent biokinetic parameters for P. putida have been calculated from continuous system data, which are most appropriate for use in continuous bioprocess applications.
Enhanced oily sludge biodegradation by a tensio-active agent isolated from Pseudomonas aeruginosa USB-CS1 by C. Rocha; C. Infante (pp. 615-619).
The biodegradation of an oily sludge is facilitated by a microbial tensio-active agent isolated from Pseudomonas aeruginosa USB-CS1. The optimal oil-in-water dispersion conditions are as follows: pH 6.5, temperature 30 °C, agitation 150 rev/min. The total hydrocarbon content shows that the biodegradation of the oily substrate mediated by the biosurfactant or by the biosurfactant–P. aeruginosa USB-CS1 complex is significantly higher after 30 days of incubation than that in other experimental conditions, by a mean of 70%. Substrate fractionation by column chromatography reveals that, if biosurfactant is present, saturated and aromatic compounds are more susceptible to microbial degradation than they are in other biodegradation systems by an average of 55% and 40% respectively. These results suggest that the stimulatory effects of the biosurfactant on the biodegradation of the oily substrate are limited over time by the loss of surface activity of the biosurfactant after 30 days of incubation.
Screening for fungi capable of removing benzo[a]pyrene in culture by K. G. Wunch; T. Feibelman; J. W. Bennett (pp. 620-624).
Some 17 strains of filamentous fungi, encompassing 13 different species, were tested for their ability to decolorize the polymeric dye R-478. Decolorization was observed with both living and dead mycelia of the three Aspergillus species tested, indicating bioadsorption, not biodegradation. With mycelia of other strains tested, the most decolorization was obtained with Marasmiellus troyanus, Pleurotus sapidus, and Pleurotus ostreatus; with extracellular filtrates, the most decolorization was observed with Laetiporus sulphureus. Parallel experiments incubating benzo[a]pyrene (B[a]P) with mycelia and filtrates showed that six of the species removed over 40% of B[a]P in comparison with HgCl2-killed controls. The highest B[a]P removal by mycelia was shown by M. troyanus (95.0%); the highest level by extracellular filtrates was shown by Hericium erinaceous (44.8%). With the exception of A. ochraceous, no products of B[a]P metabolism were detected for any of the species tested. For most species, the disappearance of B[a]P was correlated with the ability to decolorize poly R-478 ( r = 0.78 for mycelia; r = 0.74 for culture fluids). M. troyanus gave rise to more disappearance than decolorization. The removal of B[a]P by M. troyanus and Phanerochaete chrysosporium was compared over 30 days: M. troyanus gave significantly better removal in a biphasic pattern.