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  • Andersson, Erik, et al. (författare)
  • Incomplete degradation of polycyclic aromatic hydrocarbons in soil inoculated with wood rotting fungi and their effect on the indigenous soil bacteria.
  • 2003
  • Ingår i: Environmental Toxicology and Chemistry. - Society of Environmental Toxicology and Chemistry. - 1551-5028. ; 22:6, s. 1238-1243
  • Tidskriftsartikel (refereegranskat)abstract
    • Soil artificially contaminated with fluorene, phenanthrene, pyrene, and benz[a]anthracene was inoculated with the wood-rotting fungi Pleurotus ostreatus and Antrodia vaillantii. During 12 weeks of incubation, polycyclic aromatic hydrocarbon (PAH) degradation and the formation of persistent degradation products were monitored by chemical analysis. In addition, the effect on the indigenous soil bacteria was studied by plate count techniques and by measuring the concentration of bacteria-specific phospholipid fatty acids (PLFAs). In both soils inoculated with fungi, the PAH degradation was enhanced compared to the control soil without fungi. The white-rot fungus P. ostreatus accelerated the degradation rate radically the first weeks, while the effect of the brown-rot fungus was more pronounced at later stages during the 12-week study. In a soil with no amendments, the final degradation result was similar to that in the soil with added fungi, although the degradation pattern for the individual PAHs was different. Furthermore, the degradation by P. ostreatus was accompanied by an accumulation of PAH metabolites, that is, 9-fluorenone, benz[a]anthracene-7,12-dione, and two compounds identified as 4-hydroxy-9-fluorenone and 4-oxapyrene-5-one, that was not seen in the other soils. The inoculation with the white-rot fungus also had a large negative effect on the indigenous soil bacteria. This could be an important drawback of using the white-rot fungus P. ostreatus in soil bioremediation since a sequential fungal–bacterial degradation probably is needed for a complete degradation of PAHs in soil. In the soil inoculated with A. vaillantii, on the other hand, no metabolites accumulated, and no negative effects were observed on the indigenous microorganisms.
  • Arvidsson, Pär, et al. (författare)
  • Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns
  • 2002
  • Ingår i: Journal of Chromatography A. - Elsevier Science. - 0021-9673. ; 977:1, s. 27-38
  • Tidskriftsartikel (refereegranskat)abstract
    • Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylanlino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 mum in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use. (C) 2002 Published by Elsevier Science B.V.
  • Arvidsson, Pär, et al. (författare)
  • Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent
  • 2003
  • Ingår i: Journal of Chromatography A. - Elsevier. - 0021-9673. ; 986:2, s. 275-290
  • Tidskriftsartikel (refereegranskat)abstract
    • A continuous supermacroporous matrix has been developed allowing direct capture of enzyme from non-clarified crude cell homogenate at high flow-rates. The continuous supermacroporous matrix has been produced by radical co-polymerization of acrylamide, allyl glycidyl ether and N,N?-methylene-bis(acrylamide) which proceeds in aqueous solution of monomers frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix having interconnected pores of 10–100 m size. Iminodiacetic acid covalently coupled to the cryogel is a rendering possibility for immobilized metal affinity chromatographic purification of recombinant His-tagged lactate dehydrogenase, (His)6-LDH, originating from thermophilic bacterium Bacillus stearothermophilus, but expressed in Escherichia coli. The large pore size of the adsorbent makes it possible to process particulate-containing material without blocking the column. No preliminary filtration or centrifugation is needed before application of crude extract on the supermacroporous column. A total of 210 ml crude homogenate, 75 ml of it non-clarified, was processed on a single 5.0 ml supermacroporous column at flow speeds up to 12.5 ml/min without noticeable impairment of the column properties. Mechanically the cryogel adsorbent is very stable. The continuous matrix could easily be removed from the column, dried at 70 °C and kept in a dry state. After rehydration and reinsertion of the matrix into an empty column, (His)6-LDH was purified as efficiently as on the newly prepared column. The procedure of manufacturing the supermacroporous continuous cryogel is technically simple. Starting materials and initiators are cheap and available and are simply mixed and frozen under specified conditions. Altogether these qualities reveal that the supermacroporous continuous cryogels is a very interesting alternative to existing methods of protein purification from particulate-containing crude extracts
  • Bakhtiar, Shahrzad, et al. (författare)
  • Stability characteristics of a calcium-independent alkaline protease from Nesterenkonia sp.
  • 2003
  • Ingår i: Enzyme and Microbial Technology. - Elsevier. - 0141-0229. ; 32:5, s. 525-531
  • Tidskriftsartikel (refereegranskat)abstract
    • Thermodynamic stability of an alkaline protease from a new alkaliphilic Nesterenkonia sp. AL-20, was investigated and compared with that of Subtilisin Carlsberg. The amount of calcium bound to the AL-20 protease was determined to be only about 0.14 mol/mol of protease. Differential scanning calorimetry scan of the enzyme at increasing temperature showed the denaturation of the enzyme to be a two-state process with melting temperature, Tm of about 74 °C at pH 10.0, which was unaltered upon addition of calcium as well as after treatment with chelating agents. The thermodynamic parameters were nearly the same over a pH range of 7.0–10.0. Tm was reduced to 69.7 °C at pH 6.0 and 72 °C at pH 11.0. The secondary structure of the protease was unaffected during storage at 50 °C, even in the presence of 1% SDS as observed by circular dichroism. The protease activity was extremely stable in the presence of hydrogen peroxide and various sequestering agents used in detergents.
  • Balan, S, et al. (författare)
  • Metal chelate affinity precipitation of RNA and purification of plasmid DNA
  • 2003
  • Ingår i: Biotechnology Letters. - Springer. - 0141-5492. ; 25:13, s. 1111-1116
  • Tidskriftsartikel (refereegranskat)abstract
    • The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.
  • Birgisson, Hakon, et al. (författare)
  • A new thermostable alpha-L-arabinofuranosidase from a novel thermophilic bacterium
  • 2004
  • Ingår i: Biotechnology Letters. - Springer Science. - 0141-5492. ; 26:17, s. 1347-1351
  • Tidskriftsartikel (refereegranskat)abstract
    • An alpha-L-arabinofuranosidase gene was identified in a sequenced genome of a novel thermophilic bacterium, which belongs to the recently described phylum of Thermomicrobia. Amino acid sequence comparison of the enzyme (designated AraF) revealed similarity to glycoside hydrolases of family 51. The gene was cloned into Escherichia coli and its recombinant product expressed and purified. The enzyme appeared to be a hexamer. AraF was optimally active at 70degreesC (over 10 min) and pH 6 having 92% residual activity after 1 h at 70degreesC. AraF had a K-m value of 0.6 rum and V-max value of 122 U mg(-1) on p-nitrophenyl-alpha-L-arabinofuranoside. AraF was almost equally active on branched arabinan and debranched arabinan, properties not previously found in alpha-L-arabinofuranosidases in GH family 51.
  • Birgisson, Hakon, et al. (författare)
  • Cold adapted yeasts as producers of cold active polygalacturonases
  • 2003
  • Ingår i: Extremophiles. - Springer. - 1431-0651. ; 7:3, s. 185-193
  • Tidskriftsartikel (refereegranskat)abstract
    • Eight cold-adapted, polygalacturonase-producing yeasts belonging to four species were isolated from frozen environmental samples in Iceland. They were identified as Cystofilobasidium lari-marini, Cystofilobasidium capitatum, Cryptococcus macerans and Cryptococcus aquaticus species by sequence analysis of rDNA regions. Growth behavior of the isolates was investigated. All strains could grow at 2°C. Addition of glucose to pectin-containing culture medium had a repressive effect on enzyme production except for C. aquaticus, which showed increased polygalacturonase activity. Optimal temperature for enzyme production for the Cystofilobasidium strains was 14°C, while that for the Cryptococcus strains was lower. Among the isolates, C. lari-marini S3B produced highest levels of enzyme activity at pH 3.2. Preliminary characterization of the polygalacturonases in the culture supernatant showed the enzyme from Cystofilobasidium strains to be optimally active at 40°C and pH 5, and that from the Cryptococcus strains at 50°C and pH 4. The polygalacturonase from C. macerans started to lose activity after 1 h of incubation at 40°C, while that from the other strains had already lost activity at 30°C. All the strains except C. aquaticus produced isoenzymes of polyglacturonase. In addition to polygalacturonase, the Cystofilobasidium strains produced pectin lyase, C. aquaticus pectin esterase, and C. macerans pectin lyase, pectate lyase and pectin esterase.
  • Birgisson, Hakon, et al. (författare)
  • Two new thermostable alpha-L-rhamnosidases from a novel thermophilic bacterium
  • 2004
  • Ingår i: Enzyme and Microbial Technology. - Elsevier Inc.. - 0141-0229. ; 34:6, s. 561-571
  • Tidskriftsartikel (refereegranskat)abstract
    • Two new thermostable alpha-L-rhamnosidases with novel substrate hydrolysis pattern were cloned and expressed from a new thermophilic bacterium. Fragments of the two alpha-L-rhamnosidase genes, rhmA and rhmB were identified in a partially sequenced genome of the bacterium. Whole genes were recovered by amplifying flanking sequences with single specific primers and nonspecific walking primers. The recovered Genes were then cloned into Escherichia coli and their enzymes produced and purified. Both enzymes were dimers and the MW of the monomers. were 104 and 107 kDa for RhmA and RhmB, respectively. Both rhamnosidases had a temperature optimum at 70degreesC. RhmA had pH optimum at 7.9 and RhmB had a broad pH optimum of 5.0 to 6.9 and RhmA had over 50% activity in the pH interval 5.0 to 8.7 and RhmB in the pH interval 4.0 to 7.9. Both enzymes had over 20% residual activity after 24-h incubation at 60degreesC. RhmA and RhmB had K values of 0.46 and 0.66 mM and V-max values of 134 and 352 U mg(-1) respectively, on p-nitrophenyl-alpha-L-rhamnopyrano side. Both rhamnosidases were active on both alpha-1,2- and alpha-1,6-linkages to beta-D-glucoside. (C) 2004 Elsevier Inc. All rights reserved.
  • Björnsson, Lovisa, et al. (författare)
  • Evaluation of new methods for the monitoring of alkalinity dissolved hydrogen and the microbial community in anaerobic digestion
  • 2001
  • Ingår i: Water Research. - Elsevier. - 0043-1354 (print). ; 35:12, s. 2833-2840
  • Tidskriftsartikel (refereegranskat)abstract
    • New methods for spectrophotometric alkalinity measurement, dissolved hydrogen monitoring and for obtaining a fingerprint of the microbial community were evaluated as tools for process monitoring in anaerobic digestion. The anaerobic digestion process was operated at organic loading rates of 1.5, 3.0 and 4.5g volatile solids l-1d-1 and subjected to pulse loads of carbohydrate, lipid, protein and a mixed sludge substrate. The spectrophotometric alkalinity monitoring method showed good agreement with traditional titrimetric alkalinity monitoring and has the advantage of being easy to modify to on-line monitoring applications. The on-line monitoring of dissolved hydrogen gave valuable information about approaching process overload and can be a good complement to the conventional monitoring of volatile fatty acids. Changing process conditions were also reflected in the microbial fingerprint that could be achieved by partitioning in two-phase systems. The investigated methods showed potential for application in increasing our understanding of the anaerobic digestion process as well as for being applicable for monitoring in the complex environment of full-scale anaerobic digestion processes.
  • Björnsson, Lovisa, et al. (författare)
  • Evaluation of parameters for monitoring an anaerobic co-digestion process
  • 2000
  • Ingår i: Applied Microbiology and Biotechnology. - Springer. - 1432-0614 (Online) .- 0175-7598 (Print). ; 54:6, s. 844-849
  • Tidskriftsartikel (refereegranskat)abstract
    • The system investigated in this study is an anaerobic digester at a municipal wastewater treatment plant operating on sludge from the wastewater treatment, co-digested with carbohydrate-rich food-processing waste. The digester is run below maximum capacity to prevent overload. Process monitoring at present is not extensive, even for the measurement of on-line gas production rate and off-line pH. Much could be gained if a better program for monitoring and control was developed, so that the full capacity of the system could be utilised without the risk of overload. The only limit presently set for correct process operation is that the pH should be above 6.8. In the present investigation, the pH was compared with alkalinity, gas production rate, gas composition and the concentration of volatile fatty acids (VFA). Changes in organic load were monitored in the full-scale anaerobic digester and in laboratory-scale models of the plant. Gas-phase parameters showed a slow response to changes in load. The VFA concentrations were superior for indicating overload of the microbial system, but alkalinity and pH also proved to be good monitoring parameters. The possibility of using pH as a process indicator is, however, strongly dependent on the buffering capacity. In this study, a minor change in the amount of carbohydrates in the substrate had drastic effects on the buffering effect of the system.
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