Hans Kristian Kotlar

Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-l-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l−1) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

In situ compatibilization of polypropylene and poly(butylene terephthalate) polymer blends by one-step reactive extrusion

Abstract This paper concerns the in situ compatibilization of immiscible polypropylene/poly(butylene terephthalate) (PP/PBT) blends by a one-step reactive extrusion process. By one-step reactive extrusion, we are referring to functionalization of the PP with a monomer bearing a desired functional group and the subsequent interfacial reaction of this functionalized PP with the PBT, where the whole operation is carried out in a single extrusion process. Three monomers, acrylic acid (AA), maleic anhydride (MA) and glycidyl methacrylate (GMA), which are potentially reactive towards the car☐ylic and/or hydroxyl groups at the chain ends of the PBT, were melt grafted on to the PP by free-radical reactions. A comparative study showed that GMA is more effective in compatibilizing the PP/PBT blends. Optimization of chemical parameters (initial concentrations of GMA and peroxide) and processing conditions (the sequence of addition of the materials, devolatilization of residual GMA, etc.) allowed us to obtain compatibilized PP/PBT blends with a fifteen- to twentyfold improvement in elongation at break and impact strength over an uncompatibilized PP/PBT blend. The in situ compatibilization performance of this one-step reactive extrusion process was comparable with that of a classical two-step process. In this latter case, the first extrusion step was to functionalize the PP with GMA, and the second one being the interfacial reaction between the functionalized PP and the PBT.

High coverage sequencing of DNA from microorganisms living in an oil reservoir 2.5 kilometres subsurface

Microorganisms colonize a variety of extreme environments, and based on cultivation studies and analyses of PCR-amplified 16S rDNA sequences, microbial life appears to extend deep into the earth crust. However, none of these studies involved comprehensive characterizations of total DNA. Here we report results of a high-coverage DNA pyrosequencing of an apparently representative and uncontaminated sample from a deep sea oil reservoir located 2.5 km subsurface, attributing a pressure and temperature of 250 bars and 85°C respectively. Bioinformatic analyses of the DNA sequences indicate that the reservoir harbours a rich microbial community dominated by a smaller number of taxa. Comparison of the metagenome with sequences in databases indicated that there may have been contact between the oil reservoir and surface communities late in the sequence of geological events leading to oil reservoir formation. One specific gene, encoding a putative enolase, was synthesized and expressed in Escherichia coli. Enolase activity was confirmed and was found to be much more thermotolerant than for a corresponding E. coli enzyme, consistent with the conditions in the oil reservoir.

The microbial communities in two apparently physically separated deep subsurface oil reservoirs show extensive DNA sequence similarities.

It is well established that micro-organisms colonize a variety of extreme environments, including habitats like oil reservoirs deep inside the earth crust. Here, we present the results of a comparative high-coverage DNA sequencing study of metagenomes derived from two different oil reservoirs, both located about 2.5 km subseafloor below the Norwegian Sea. A previously reported bioinformatic analysis of DNA sequence data derived from one of the reservoirs (Well I) indicated that the community is dominated by bacterial species with a smaller fraction of Archaea. Here, we report results of a similar analysis from another reservoir (Well II) located in the same geographical area, however, according to available geological knowledge lacking direct physical contact with Well I. Interestingly, the Well II community is largely dominated by Archaea with a subordinate fraction of Bacteria. Comparison of the two datasets showed that large fractions of the sequences are extremely similar, both with respect to identity (typically above 98%) and gene organization. We therefore conclude that both wells contain essentially the same organisms, but in different relative abundances. Assuming that the communities have been distinct for long timescales because of physical separation, the results also indicate that microbial growth in the reservoirs is extremely slow.

The structure of a tetrameric α-carbonic anhydrase from Thermovibrio ammonificans reveals a core formed around intermolecular disulfides that contribute to its thermostability

Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificans α-carbonic anhydrase (TaCA) has been expressed in Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized α-carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold.

Ralstonia sp. U2 naphthalene dioxygenase and Comamonas sp. JS765 nitrobenzene dioxygenase show differences in activity towards methylated naphthalenes

Methylsubstituted naphthalenes constitute a significant part of light gas oil fractions (LGO). These are toxic compounds with low fuel value, and can potentially be enzymatically modified to increase the fuel value and at the same time reduce toxicity. The first step in the biodegradation of naphthalene involves dioxygenation of the aromatic ring catalysed by naphthalene dioxygenase (NDO). Here we show that recombinantly produced NDO from Ralstonia sp. U2 and the related nitrobenzene dioxygenase (NBDO) from Comamonas sp. JS765 can use several mono-, di-, tri-, and tetramethylated naphthalenes as substrates. For the majority of the substrates both enzymes catalyse the formation of a mixture of mono- and dioxygenated products, and it is only dioxygenated products that are likely to be processed further, leading to ring cleavage. In some cases, like for 1-methylnaphthalene, NDO mainly generates the monooxygenated form, while with NBDO, the dioxygenated form dominates. In other cases, as for 1,4-dimethylnaphthalene, the monooxygenated product dominates with NDO, whereas NBDO generates similar amounts of both forms. Presumably, the best future strategy for bioconversion of methylated naphthalenes in LGO is to develop engineered enzyme that are optimised with respect to the specific composition of naphthalene derivatives found in a given product.

Polypropylene–phenol formaldehyde‐based compatibilizers. III. Application in PP/PBT and PP/PPE blends

A new class of compatibilizers suitable for blends or alloys of polypropylene (PP) and engineering polymers having aromatic residues or functionality complimentary to hydroxyl were evaluated in blends of isotactic PP and poly(butylene terephthalate) (PBT), and poly(phenylene ether) (PPE) PP-based blends with 10-30 wt % PBT or PPE were studied. From response modeling of the PP/PBT and PP/PPE blends, it was evident that the viscosity ratio among the blend component, compatibilizer content, and PBT or PPE content were important for the final blend properties. Impact strength was observed to be the most sensitive response to blend compatibilization. The PP-g-PF compatibilizer was observed to be more efficient in blends of PBT than of PPE. The main reason for that was the availability of reactive end-groups in the case of PBT, making covalent bonding between the compatibilizer and PBT possible.

Deep Subsurface Oil Reservoirs as Poly-extreme Habitats for Microbial Life. A Current Review

Oil reservoirs located deep within the earth crust represent one of the most challenging environments for life, usually providing combinations of high temperatures and pressures, as well as high concentrations of salts, heavy metals, and organic solvents. Organisms thriving in such environments, therefore, have to be truly poly-extremophiles, adapted to conditions otherwise very hostile to life. In spite of this, research carried out in many groups worldwide throughout the past decades has revealed that deep subsurface oil reservoirs indeed are populated by diverse consortia of poly-extremophilic Bacteria and Archaea. Numerous sites on all continents have been sampled in search for novel species and strains to describe and compare microbial consortia and to understand biological processes that might occur in response to and possibly interfere with an efficient oil production. In addition, the special adaptations of oil reservoir microbes to their extreme environments have rendered them highly attractive for bioprospecting approaches for novel enzymes and metabolites with potential industrial value.

Microbial communities of a complex high-temperature offshore petroleum reservoir

In this paper, the microbial communities of a complex high-temperature (appr. 85°) offshore petroleum reservoir are described as part of improving the understanding of the biogeochemical processes in the reservoir. Based on molecular biology studies, the results revealed that the microbial communities differed between samples from producing wells and the oil/water production facility of the reservoir, although a few types of thermophilic microbes appeared in both environments. The microbial communities of the different producing wells could be separated into three groups, and the target bacteria related to the genera Halomonas, Arcobacter or Pseudomonas seemed to be associated with these groups. Thus, different microbial communities were detected in this reservoir, and further investigations will clarify if specific target microbes may be used for the identification of different oil qualities or other reservoir characteristics. [Received: November 4, 2007; Accepted: January 10, 2008]

Polypropylene–phenol formaldehyde‐based compatibilizers. II. Application in PP/PA6 75/25 (wt/wt) blends

A new class of compatibilizers suitable for blends or alloys of polypropylene and engineering polymers having aromatic residues or functionality complimentary to hydroxyl were evaluated in blends of isotatctic polypropylene (PP) and polyamide 6 (PA6). The compatibilizer consisted of a PP part with a phenol formaldehyde (PF) polymer grafted onto it. In this study, various combinations of the polymer parameter of each compatibilizer building block were examined. Based on the same loading, the compatibilizer with low molecular weight PP and high content of high molecular weight PF was observed to be the most efficient. A compatibilizer content of up to 7.5% by weight gave significant reduction in the average particle size of the dispersed PA phase. Similarly, corresponding improvements in the mechanical properties were observed as the average particle size was reduced. For some of the blends, more than additive improvement in the mechanical properties were achieved.