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ABSTRACT: Plant cell wall structures represent a barrier in the biodegradation process to produce biogas for combustion and energy production. Consequently, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required. Here, we show that natural activated zeolites (i.e. trace metal activated zeolites) represent eminently suitable mineral microhabitats and potential carriers for immobilisation of microorganisms responsible for anaerobic hydrolysis of biopolymers stabilising related bacterial and methanogenic communities. A strategy for comprehensive analysis of immobilised anaerobic populations was developed that includes the visualisation of biofilm formation via scanning electron microscopy and confocal laser scanning microscopy, community and fingerprint analysis as well as enzyme activity and identification analyses. Using SDS polyacrylamide gel electrophoresis, hydrolytical active protein bands were traced by congo red staining. Liquid chromatography/mass spectroscopy revealed cellulolytical endo- and exoglucanase (exocellobiohydrolase) as well as hemicellulolytical xylanase/mannase after proteolytic digestion. Relations to hydrolytic/fermentative zeolite colonisers were obtained by using single-strand conformation polymorphism analysis (SSCP) based on amplification of bacterial and archaeal 16S rRNA fragments. Thereby, dominant colonisers were affiliated to the genera Clostridium, Pseudomonas and Methanoculleus. The specific immobilisation on natural zeolites with functional microbes already colonising naturally during the fermentation offers a strategy to systematically supply the biogas formation process responsive to population dynamics and process requirements.
Applied Microbiology and Biotechnology 02/2013; · 3.42 Impact Factor
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D Ribitsch,
S Heumann,
W Karl,
J Gerlach,
R Leber,
R Birner-Gruenberger,
K Gruber,
I Eiteljoerg,
P Remler,
P Siegert,
J Lange,
K H Maurer,
G Berg, G M Guebitz,
H Schwab
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ABSTRACT: A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45°C. Specific activity of StmPr2 determined with suc-L-Ala-L-Ala-L-Pro-l-Phe-p-nitroanilide as the substrate was 17±2U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.
Journal of biotechnology 01/2012; 157(1):140-7. · 2.88 Impact Factor
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W Schloegl,
A Klein,
R Fürst,
U Leicht,
E Volkmer,
M Schieker,
S Jus, G M Guebitz,
I Stachel,
M Meyer,
M Wiggenhorn,
W Friess
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ABSTRACT: In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) for microbial transglutaminase (mTG) from Streptomyces mobaraensis to overcome the lack of a quantification method for mTG. We further performed a detailed follow-on-analysis of insoluble porcine collagen type I enzymatically modified with mTG primarily focusing on residuals of mTG. Repeated washing (4 ×) reduced mTG-levels in the washing fluids but did not quantitatively remove mTG from the material (p < 0.000001). Substantial amounts of up to 40% of the enzyme utilized in the crosslinking mixture remained associated with the modified collagen. Binding was non-covalent as could be demonstrated by Western blot analysis. Acidic and alkaline dialysis of mTG treated collagen material enabled complete removal the enzyme. Treatment with guanidinium chloride, urea, or sodium chloride was less effective in reducing the mTG content.
European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 11/2011; 80(2):282-8. · 3.15 Impact Factor
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ABSTRACT: The colonisation of activated zeolites (i.e. clinoptilolites) as carriers for microorganisms involved in the biogas process was investigated. Zeolite particle sizes of 1.0-2.5mm were introduced to anaerobic laboratory batch-cultures and to continuously operated bioreactors during biogas production from grass silage. Incubation over 5-84 days led to the colonisation of zeolite surfaces in small batch-cultures (500 ml) and even in larger scaled and flow-through disturbed bioreactors (28 l). Morphological insights were obtained by using scanning electron microscopy (SEM). Single strand conformation polymorphism (SSCP) analysis based on amplification of bacterial and archaeal 16S rRNA fragments demonstrated structurally distinct populations preferring zeolite as operational environment. via sequence analysis conspicuous bands from SSCP patterns were identified. Populations immobilised on zeolite (e.g. Ruminofilibacter xylanolyticum) showed pronounced hydrolytic enzyme activity (xylanase) shortly after re-incubation in sterilised sludge on model substrate. In addition, the presence of methanogenic archaea on zeolite particles was demonstrated.
Bioresource technology 03/2011; 102(6):4353-9. · 4.25 Impact Factor
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ABSTRACT: Biogas from agricultural biomass and residues is a valuable source of renewable energy. However, recalcitrant plant cell structures represent a barrier in the fermentative biodegradation process in single- and two-stage reactors. Therefore, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required amongst others in order to optimise the fermentation efficiency and to increase methane yields. Here we show a new strategy for the enhancement of biogas production from hemicellulose-rich substrates. Hemicellulolytic populations from a common biogas fermenter consortium were successively enriched in batch-cultures using a synthetic medium containing xylan powder as single carbon source under anaerobic mesophilic conditions. Enriched hemicellulolytic bacteria were immobilised on trace metal activated zeolite to ensure a stable storage and easy application. Xylanase activity increased continuously during subsequent enrichment cycles by up to 162%. In batch-culture experiments we were able to observe an increase of methane by 53% compared to controls without additionally introduced microorganisms immobilised on zeolite. Specific enrichment of hemicellulolytic bacteria during the process was confirmed by using single strand conformation polymorphism (SSCP) analysis based on amplification of the eubacterial 16S rDNA fragments. Using sequence analysis conspicuous bands from SSCP patterns could be identified as belonging to the groups Bacteroides sp., Azospira oryzae (Dechlorosoma sp.) as well as to a wide spectrum of diverse species within the order of Clostridiales (Firmicutes).
Water Research 11/2009; 44(6):1970-80. · 4.86 Impact Factor
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ABSTRACT: Pseudomonas putida GG04 and Bacillus SF have been successfully incorporated into an explosive formulation to enhance biotransformation of TNT residues and/or explosives which fail to detonate due to technical faults. The incorporation of the microorganisms into the explosive did not affect the quality of the explosive (5 years storage) in terms of detonation velocity while complete biotransformation of TNT moieties upon transfer in liquid media was observed after 5 days. The incorporated microorganisms reduced TNT sequentially leading to the formation of hydroxylaminodinitrotoluenes (HADNT), 4-amino-2,6-dinitrotoluenes; 2-amino-4,6-dinitrotoluenes, different azoxy compounds; 2,6-diaminonitrotoluenes (2,4-DAMNT) and 2,4-diaminonitrotoluenes (2,6-DAMNT). However, the accumulation of AMDNT and DAMNT (major dead-end metabolites) was effectively prevented by incorporating guaiacol and catechol during the biotransformation process.
Journal of hazardous materials 11/2008; 165(1-3):285-90. · 4.14 Impact Factor
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ABSTRACT: The potential of accumulation of lipids by Lipomyces starkeyi when grown on sewage sludge was assessed. On a synthetic medium, accumulation of lipids strongly depended on the C/N ratio. The highest content of lipids was measured at a C/N-ratio of 150 with 68% lipids of the dry matter while at a C/N-ratio of 60 only 40% were accumulated. Within a pH range from 5.0 to 7.5 the highest lipid accumulation was found at pH 5.0 while the highest yield per litre was pH 6.5. Although sewage sludge had no inhibitory effects on growth or accumulation on L. starkeyi when added to synthetic medium, there was no significant growth on untreated sewage sludge. However, pretreatment of sludge by alkaline or acid hydrolysis, thermal or ultrasonic treatment lead to accumulation of lipids by L. starkeyi with highest values of 1 g L(-1) obtained with ultrasound pre-treatment. Based on the content of free fatty acids and phosphorus, lipids accumulated from sewage sludge could serve as a substrate for the production of biodiesel.
Bioresource Technology 06/2008; 99(8):3051-6. · 4.98 Impact Factor
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ABSTRACT: Contaminating microorganisms such as Actinomycetes, Alicyclobacillus, and Chlostridium can generate off-flavors in apple juices. Such bacterial metabolites represent, besides phenol types such as guaiacol and 2,6-dibromophenol, a broad range of other chemicals, for example, geosmin, 2-methylisoborneol, or alpha-terpineol. A laccase from Trametes hirsuta was purified, immobilized, and applied for the selective elimination of off-flavor substances in apple juice caused by microbial contamination. The evaluation using GC-MS showed that enzymatic treatment could reduce the amount of guaiacol and 2,6-dibromophenol in apple juice significantly by 99 and 52%, respectively. Upon addition of mediators, the degradation could be increased and the spectrum of substrates extented. Furthermore, commercial apple juices spiked with off-flavors were treated in a continuous-flow reactor and tested by sensory evaluation.
Journal of Agricultural and Food Chemistry 05/2008; 56(7):2485-9. · 2.82 Impact Factor
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ABSTRACT: A customer- and environment-friendly method for the decolorization azo dyes was developed. Azoreductases could be used both to bleach hair dyed with azo dyes and to reduce dyes in vat dyeing of textiles. A new reduced nicotinamide adenine dinucleotide-dependent azoreductase of Bacillus cereus, which showed high potential for reduction of these dyes, was purified using a combination of ammonium sulfate precipitation and chromatography and had a molecular mass of 21.5 kDa. The optimum pH of the azoreductase depended on the substrate and was within the range of pH 6 to 7, while the maximum temperature was reached at 40 degrees C. Oxygen was shown to be an alternative electron acceptor to azo compounds and must therefore be excluded during enzymatic dye reduction. Biotransformation of the azo dyes Flame Orange and Ruby Red was studied in more detail using UV-visible spectroscopy, high-performance liquid chromatography, and mass spectrometry (MS). Reduction of the azo bonds leads to cleavage of the dyes resulting in the cleavage product 2-amino-1,3 dimethylimidazolium and N approximately 1 approximately ,N approximately 1 approximately -dimethyl-1,4-benzenediamine for Ruby Red, while only the first was detected for Flame Orange because of MS instability of the expected 1,4-benzenediamine. The azoreductase was also found to reduce vat dyes like Indigo Carmine (C.I. Acid Blue 74). Hydrogen peroxide (H(2)O(2)) as an oxidizing agent was used to reoxidize the dye into the initial form. The reduction and oxidation mechanism of Indigo Carmine was studied using UV-visible spectroscopy.
Applied Microbiology and Biotechnology 12/2007; 77(2):321-7. · 3.42 Impact Factor
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ABSTRACT: In this paper a new enzymatic process direction is described for obtaining machine washable wool with acceptable quality. In general, application of protease enzyme technology in wool processing results in considerable loss of tensile strength by diffusion of the enzyme into the interior of wool fibers. To overcome this disadvantage enzymatic activity has been more targeted to the outer surface of the scales by improving the susceptibility of the outer surface scale protein for proteolytic degradation. This has been realized by a pretreatment of wool with hydrogen peroxide at alkaline pH in the presence of high concentrations of salt.
Biotechnology Letters 06/2006; 28(10):711-6. · 1.68 Impact Factor
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ABSTRACT: From a screening for the production of new proteases specific for cuticle scales, Beauveria brongniartii was selected producing an alkaline Ca(++) dependent protease. The purified had a molecular weight of 27 kDa and a pI value of 8.0. Substrate specificities of model substrates (wool with partially removed cuticles treated with SDS) were analyzed by protein release, dissolved organic carbon (DOC) and nitrogen analysis. The C/N ratio of released material turned out to be a good parameter to determine the site of action of proteases on fibres. Compared to other enzymes, the fungal protease preferentially hydrolyzed cuticle scales and has thus a potential for anti-shrinking pre-treatment of wool fabrics.
Biotechnology Letters 06/2006; 28(10):703-10. · 1.68 Impact Factor
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ABSTRACT: Laccases could prevent fabrics and garments from re-deposition of dyes during washing and finishing processes by degrading the solubilized dye. However, laccase action must be restricted to solubilized dye molecules thereby avoiding decolorization of fabrics. Chemical modification of enzymes can provide a powerful tool to change the adsorption behaviour of enzymes on water insoluble polymers. Polyethylene glycol (PEG) was covalently attached onto a laccase from Trametes hirsuta. Different molecular weights of the synthetic polymer were tested in terms of adsorption behaviour and retained laccase activity. Covalent attachment of PEG onto the laccase resulted in enhanced enzyme stability while with increasing molecular weight of attached PEG the substrate affinity for the laccase conjugate decreased. The activity of the modified laccases on fibre bound dye was drastically reduced decreasing the adsorption of the enzyme on various fabrics. Compared to the 5 kDa PEG laccase conjugate (K/S value 47.60) the K/S value decreased much more (47.96-46.35) after the treatment of dyed cotton fabrics with native laccase.
Biotechnology Letters 06/2006; 28(10):741-7. · 1.68 Impact Factor
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ABSTRACT: A new strain of Trametes hirsuta was found to oxidize various cotton flavonoids. Here we show that laccases of this organism were responsible for oxidation
of the flavonoids morin, luteolin, rutin and quercetin. Out of two laccases produced by T. hirsuta (60.7 and 51.0kDa) the more prominent 60.7kDa laccase was purified and showed K
m and k
cat values of 75.5, 20.9 and 49.4μM and 72.5, 96.3 and 32.7s−1, hours on ABTS, syringaldazide and DMP, respectively. Pretreatment of cotton with the T. hirsuta laccase resulted in a whiteness increase of 8.5%.
Environmental Chemistry Letters 10/2005; 3(2):66-69. · 1.88 Impact Factor
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ABSTRACT: Treatment of effluents containing phenols such as textile dyes with fungal laccases is usually limited to the acid to neutral
pH range and moderate temperatures. Here we demonstrate for the first time that spore-bound laccases which are stable at high
temperatures and pH values can be used for phenolic dye decolorisation. Laccase containing spores from Bacillus SF were immobilized on alumina pellets. Both immobilized and free spores were able to completely decolorize the common textile
dyes Mordant Black 9, Mordant Brown 96/Mordant Brown 15, and Acid Blue 74 within 90min of incubation time and decolorized
solutions were successfully used in re-dyeing.
Environmental Chemistry Letters 01/2005; 3(2):74-77. · 1.88 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Biogas from agricultural biomass and residues is a valuable source of renewable energy. However, recalcitrant plant cell structures represent a barrier in the fermentative biodegradation process in single- and two-stage reactors. Therefore, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required amongst others in order to optimise the fermentation efficiency and to increase methane yields. Here we show a new strategy for the enhancement of biogas production from hemicellulose-rich substrates. Hemicellulolytic populations from a common biogas fermenter consortium were successively enriched in batch-cultures using a synthetic medium containing xylan powder as single carbon source under anaerobic mesophilic conditions. Enriched hemicellulolytic bacteria were immobilised on trace metal activated zeolite to ensure a stable storage and easy application. Xylanase activity increased continuously during subsequent enrichment cycles by up to 162%. In batch-culture experiments we were able to observe an increase of methane by 53% compared to controls without additionally introduced microorganisms immobilised on zeolite. Specific enrichment of hemicellulolytic bacteria during the process was confirmed by using single strand conformation polymorphism (SSCP) analysis based on amplification of the eubacterial 16S rDNA fragments. Using sequence analysis conspicuous bands from SSCP patterns could be identified as belonging to the groups Bacteroides sp., Azospira oryzae (Dechlorosoma sp.) as well as to a wide spectrum of diverse species within the order of Clostridiales (Firmicutes).
Water Research.
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[show abstract]
[hide abstract]
ABSTRACT: The colonisation of activated zeolites (i.e. clinoptilolites) as carriers for microorganisms involved in the biogas process was investigated. Zeolite particle sizes of 1.0–2.5 mm were introduced to anaerobic laboratory batch-cultures and to continuously operated bioreactors during biogas production from grass silage. Incubation over 5–84 days led to the colonisation of zeolite surfaces in small batch-cultures (500 ml) and even in larger scaled and flow-through disturbed bioreactors (28 l). Morphological insights were obtained by using scanning electron microscopy (SEM). Single strand conformation polymorphism (SSCP) analysis based on amplification of bacterial and archaeal 16S rRNA fragments demonstrated structurally distinct populations preferring zeolite as operational environment. via sequence analysis conspicuous bands from SSCP patterns were identified. Populations immobilised on zeolite (e.g. Ruminofilibacter xylanolyticum) showed pronounced hydrolytic enzyme activity (xylanase) shortly after re-incubation in sterilised sludge on model substrate. In addition, the presence of methanogenic archaea on zeolite particles was demonstrated.
Bioresource Technology.
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[show abstract]
[hide abstract]
ABSTRACT: Pseudomonas putida GG04 and Bacillus SF have been successfully incorporated into an explosive formulation to enhance biotransformation of TNT residues and/or explosives which fail to detonate due to technical faults. The incorporation of the microorganisms into the explosive did not affect the quality of the explosive (5 years storage) in terms of detonation velocity while complete biotransformation of TNT moieties upon transfer in liquid media was observed after 5 days. The incorporated microorganisms reduced TNT sequentially leading to the formation of hydroxylaminodinitrotoluenes (HADNT), 4-amino-2,6-dinitrotoluenes; 2-amino-4,6-dinitrotoluenes, different azoxy compounds; 2,6-diaminonitrotoluenes (2,4-DAMNT) and 2,4-diaminonitrotoluenes (2,6-DAMNT). However, the accumulation of AMDNT and DAMNT (major dead-end metabolites) was effectively prevented by incorporating guaiacol and catechol during the biotransformation process.
Journal of Hazardous Materials.
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[show abstract]
[hide abstract]
ABSTRACT: Coupling of 3-(3-tert-butyl-4-hydroxyphenyl) propionic acid methylester to 1H-benzotriazole using a laccase from Trametes hirsuta was studied. The potentially resulting coupling product Tinuvin 1130 is an important UV-absorber used in polymer based materials. Oxidation of the phenol by the laccase led to homomolecular coupling reactions while the laccase did not attack 1H-benzotriazole. Due to the homomolecular reaction of the phenol in the presence of laccase coupling of phenol and 1H-benzotriazole was only observed when 1H-benzotriazole was applied in four-fold molar excess. The reaction was monitored by UV/vis spectroscopy, TLC and MS (ion trap) analysis. Coupling of 1H-benzotriazole took place in ortho position according to the postulated mechanism.
Enzyme and Microbial Technology.
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ABSTRACT: A new customer and environmental friendly method of hair bound dye decolouration was developed. Biotransformation of the azo-dyes Flame Orange and Ruby Red was studied using different oxidoreductases. The pathways of azo dye conversion by these enzymes were investigated and the intermediates and metabolites were identified and characterised using UV–vis spectroscopy, high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Laccase from Pycnoporus cinnabarinus, manganese peroxidase (MnP) from Nematoloma frowardii and the novel Agrocybe aegerita peroxidase (AaP) were found to use a similar mechanism to convert azo dyes. They N-demethylated the dyes and concomitantly polymerized them to some extent. On the other hand the mechanism for cleavage of the azo bond by azo-reductases of Bacillus cereus and B. subtilis was based on reduction of the azo bond at the expense of NAD(P)H.
Enzyme and Microbial Technology.
