Olle Holst

Influence of Sulfide and Temperature on Species Composition and Community Structure of Hot Spring Microbial Mats

ABSTRACT In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80°C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90°C, and a Chloroflexusmat is formed at 65 to 70°C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and oneChloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in theChloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter−1) below 70°C, whereasAquificales were dominant in the high-sulfide spring (12 mg liter−1) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.

Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer

SummaryThe use of isopropyl-β-d-thiogalactoside (IPTG) for induction of the lac-promoter in small-scale cultivations is well established. However, for large-scale microbiological processes the cost of this inducer is a severe limitation. Here is described a method by which lactose is used as inducer of the lac promoter with the same efficiency as that of IPTG. It was found that after growth on glucose the time of the addition of lactose is important for the quality of induction. the resulting yield of the recombinant protein increased when lactose was added to the culture if the glucose concentration was rather low. By careful monitoring of the glucose level in the fermentation, using a biosensor, it was possible to add the inducer when the carbon source was nearly depleted. Using Escherichia coli BL21 (pET3), in which was cloned the main antigen coat protein of the foot and mouth disease virus, induction of the gene led to expression of the target protein at a level exceeding 20% of the total cell protein.

Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na(+) and Ca(2+) ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and beta-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.

The effect of yoghurt culture on the survival of probiotic bacteria in oat-based, non-dairy products

The survival of the probiotic strains Lactobacillus reuteri ATCC 55730, Lactobacillus acidophilus DSM 20079 and Bifidobacterium bifidum DSM 20456, all of human origin, were investigated in three different oat-based, non-dairy products (Adavena M40, MG20 and G40). The products were fermented by the three strains with and without the presence of a commercial yoghurt culture (V2). Samples were stored at 6 degreesC up to 30 days. In general, the oat-based products were shown to be a suitable support for these intestinal bacteria. L. reuteri ATCC 55730 had the highest viability in all of the products investigated. After 30 days the cell viability of L. reuteri ATCC 55730 was 10(8) CFU ml(-1) in all three oat-based products. Lower viability was seen when the strains were grown in the presence of the yoghurt culture compared to when they were grown as pure cultures. These products also exhibited a lower pH value in comparison to products fermented with the pure cultures. The milisation of the main fermentable carbohydrates in the products varied across the bacterial strains. A decrease in beta-glucan content was seen for the products fermented by the B. bifidum DSM 20456 strain. This work shows that these oat-bases with different mono- and disaccharide composition can be used to support the growth of human intestinal bacteria and also maintain high cell viability during cold storage.

Adsorbents for extractive bioconversion applied to the acetone-butanol fermentation

SummaryFour different polymeric resins were tested as adsorbents in extractive bioconversion applied to the fermentative production of acetone and butanol by Clostridium acetobutylicum. The polymers were tested for their ability to adsorb butanol from pure solutions, and fermentation broths. Furthermore, the effect on the fermentability of the media was tested. The pH was increased to prevent adsorption of intermediates such as acetic and butyric acids. Bonopore, the polymer giving the best adsorption pattern with no undesirable effects, was tested in repeated batch cultures with C. acetobutylicum.

Oxygenation of immobilized cells using hydrogen-peroxide; a model study of Gluconobacter oxydans converting glycerol to dihydroxyacetone

SummaryThe possibility of using hydrogen-peroxide as an oxygen source in immobilized whole cell systems was investigated.The conversion of glycerol to dihydroxyacetone performed by Ca-alginate immobilized Gluconobacter oxydans was used as a model system. The influence of basic parameters, such as pH, temperature and concentration of hydrogen-peroxide, on the conversion are reported. Continuous production using a packed bed could be substantially improved by using hydrogen-peroxide as a source of oxygen. The productivity was increased almost twenty-fold in the reactor. However, hydrogen-peroxide also caused deactivation of the oxidizing of the cells.

Thermostable xylanolytic enzymes from Rhodothermus marinus grown on xylan

The thermophilic eubacterium Rhodothermus marinus was cultivated in a fermentor and studied with respect to activities of induced xylanolytic enzymes. Growth in the fermentor on xylan occurred with a maximum specific growth rate of 0.43 h−1 for a batch culture. The final cell concentration was 4 g cell dry weight (CDW)/l for cells grown on xylan compared to 2 g CDW/l for cells grown without xylan in the cultivation medium. At least two xylanolytic enzymes, endo-1,4-β-xylanase and xylan 1,4-β-xylosidase, were secreted into the culture medium when cells were cultivated on xylan. Of the three cellulolytic enzymes tested for activity, β-glucosidase activity was in the range of the xylanolytic enzyme activities whereas cellulose-1,4-β-cellobiosidase and cellulase activities were hardly detectable. The expression of endo-1,4-β-xylanase activities during cultivation indicates the existance of more than one xylanase in R. marinus. This is also observed in fractions from gel filtration. The xylanolytic enzymes are heat-stable. At 90°C and at pH 7.0 the half-life of the endo-1,4-β-xylanase was about 14 h and that of xylan 1,4-β-xylosidase was 45 min.

Oxygen supply to immobilized cells: 2. Studies on a coimmobilized algae—bacteria preparation with in situ oxygen generation

A coimmobilized mixed culture of algae, Chlorella pyrenoidosa, and bacteria, Gluconobacter oxydans, has been studied. The conversion of glycerol to dihydroxyacetone(1,3-dihydroxy-2-propanone), catalysed by the bacteria, was used to indicate the oxygen supply in the immobilized preparation. The oxygen produced by the algae in the coimmobilized preparation was used by the bacteria more effectively than when the cells were immobilized separately and mixed within the reactor. A preparation consisting of only bacteria and no algae was much less effective. The coimmobilized preparation was used in the continuous production of dihydroxyacetone for six days without any significant loss of activity.

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

Abstract A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C.

Cloning and sequence of a thermostable multidomain xylanase from the bacterium Rhodothermus marinus.

The gene (xyn1) encoding a Rhodothermus marinus xylanase has been cloned and expressed in Escherichia coli. The gene comprises 5 different domains in an unusual combination. The cellulose binding domains (CBDs) encoded by xyn1 are repeated in tandem at the N-terminus and show similarity with the CBD family IV. The xyn1-gene is the first example encoding a CBD family IV in combination with a xylan hydrolyzing catalytic domain of the glycosyl hydrolase family 10.