Molecular phylogeny of Archaea in boreal forest soil, freshwater and temperate estuarine sediment

Suurin osa luonnossa havaitsemistamme mikrobeista on sellaisia, joita emme edelleenkään osaa kasvattaa laboratorio-oloissa, vaikka tietomme mikrobien monimuotoisuudesta paranevat koko ajan. Luonnontilaisen mikrobieliöstön kokoonpano eri ympäristöissä on paljolti epäselvä ja ymmärrämme vielä hyvin puutteellisesti mikrobien ekologiaa ja niiden rooleja eliöyhteisöissä. Nykyaikaiset molekulaariset tutkimusmenetelmät auttavat selvittämään mikrobien monimuotoisuutta kokonaisvaltaisesti ja nopeasti. Ympäristöstä kemiallisesti puhdistetut ribosomaalista RNA:ta koodaavat geenit edustavat periaatteessa kaikkia eliöyhteisön geneettisesti toisistaan poikkeavia eliöitä. Niistä voidaan valikoida halutut genomit jatkotutkimuksia varten. Uusien menetelmien käyttö on tuonut esiin sen merkittävän seikan, että "tavanomaisten" elinympäristöjen eliöyhteisöihin kuuluu suuri joukko entuudestaan tuntemattomia arkkieliöitä. Aiemmin kuviteltiin, että arkkieliöt asuttavat vain sellaisia "epätavallisia" tai "äärimmäisiä" elinympäristöjä, joita luonnehtii joku seuraavista ominaisuuksista: hyvin korkea lämpötila, korkea suolapitoisuus, korkea happamuus tai emäksisyys, hapettomuus. Tutkijat ovat viimeisen noin kymmenen vuoden aikana osoittaneet, että arkkieliöt asuttavat hyvin monenlaisia kylmän ja lauhkean vyöhykkeen ympäristöjä, yhtä hyvin maaperää kuin suolaisen ja makean veden pohjaa tai pintakerroksia. Nämä löydöt ovat avanneet uuden alun arkkieliöiden tutkimukselle, erityisesti sen selvittämiselle, mitkä ovat niiden fysiologiset ja ekologiset roolit monimuotoisissa mikrobiyhteisöissä. Tämä väitöskirja kuvaa entuudestaan tuntemattomien arkkieliöiden löytymistä havumetsävyöhykkeen metsämaasta. Arkkieliöitä löytyi myös lauhkean vyöhykkeen vuorovesialueelta, murtoveden huuhtelemasta pohjasta. Nämä löydöt ovat perustavalaatuisia vuorovesialueen eliöyhteisöjen ymmärtämiseksi. Suomalaisen metsäjärven vedestä määritettiin molempien arkkieliöiden pääryhmien - tieteellisiltä nimiltään Crenarchaeota ja Euryarchaeota - edustajia. Euryarchaeota-ryhmän edustajia voitiin havainnoida myös fluoresenssi-mikroskopoinnilla. Löydöt viittaavat siihen, että arkkieliöillä on oma biogeokemiallinen roolinsa makeanveden ravintoketjujen hiilen käytössä. Tässä työssä määritetyt uudet arkkieliöiden genomien nukleotidisekvenssit on toimitettu ARB-tietokantaan, jonka kasvava vertailuaineisto edelleen parantaa uusien arkkieliösekvenssien analyysiä ja auttaa hybridisaatiokoetinten ja polymeraasiketjureaktioalukkeiden suunnittelussa ja arvioinnissa. Tässä väitöskirjassa esitellyt tulokset yhdessä lukuisien vesi-, maaperä- ja muiden ympäristöjen arkkieliöitä käsittelevien julkaisujen kanssa osoittavat, että arkkieliöt asuttavat monia erilaisia elinympäristöjä ja että ne ovat ekologisesti paljon menestyneempiä, kuin tieteenalalla on kuviteltu. Voimme olettaa, että heti kun joitain näistä eliöistä onnistutaan kasvattamaan ja ylläpitämään laboratorio-oloissa, niiden joukosta löydetään aivan uusia, entuudestaan tuntemattomia fysiologisia fenotyyppejä, jotka avaavat mielenkiintoisia näkymiä aineenvaihdunnan ja perinnöllisten ominaisuuksien tutkimukselle

Methanogenic communities in permafrost-affected soils of the Laptev Sea coast, Siberian Arctic, characterized by 16S rRNA gene fingerprints

Permafrost environments in the Arctic are characterized by extreme environmental conditions that demand a specific resistance from microorganisms to enable them to survive. In order to understand the carbon dynamics in the climate-sensitive Arctic permafrost environments, the activity and diversity of methanogenic communities were studied in three different permafrost soils of the Siberian Laptev Sea coast. The effect of temperature and the availability of methanogenic substrates on CH4 production was analysed. In addition, the diversity of methanogens was analysed by PCR with specific methanogenic primers and by denaturing gradient gel electrophoresis (DGGE) followed by sequencing of DGGE bands reamplified from the gel. Our results demonstrated methanogenesis with a distinct vertical profile in each investigated permafrost soil. The soils on Samoylov Island showed at least two optima of CH4 production activity, which indicated a shift in the methanogenic community from mesophilic to psychrotolerant methanogens with increasing soil depth. Furthermore, it was shown that CH4 production in permafrost soils is substrate-limited, although these soils are characterized by the accumulation of organic matter. Sequence analyses revealed a distinct diversity of methanogenic archaea affiliated to Methanomicrobiaceae, Methanosarcinaceae and Methanosaetaceae. However, a relationship between the activity and diversity of methanogens in permafrost soils could not be shown

Description of two biovars in the Rhizobium galegae species: biovar orientalis and biovar officinalis

Summary Twenty-six Rhizobium galegae strains, representing the center of origin of the host plants Galega orientalis and G. officinalis as well as other geographic regions, were used in a polyphasic analysis of the relationships of R. galegae strains. Phage typing, lipopolysaccharide (LPS) profiling, pulsed field gel electrophoresis (PFGE) profiling and rep-PCR (use of repetitive sequences as PCR primers for genomic fingerprinting) with REP and ERIC primers investigated nonsymbiotic properties, whereas plasmid profiling and hybridisation with a nif gene probe, and with nodB, nodD, nod box and an IS sequence from the symbiotic region as probes, were used to reveal the relationships of symbiotic genes. The results were used in pairwise calculations of distances between the strains, and the distances were visualised as a dendrogram. Indexes of association were compared for all tests pooled, and for chromosomal tests and symbiotic markers separately, to display the input of the different categories of tests on the grouping of the strains. Our study shows that symbiosis related genetic traits in R. galegae divide strains belonging to the species into two groups, which correspond to strains forming an effective symbioses with G. orientalis and G. officinalis respectively. We therefore propose that Rhizobium galegae strains forming an effective symbiosis with Galega orientalis are called R. galegae bv. orientalis and strains forming an effective symbiosis with Galega officinalis are called R. galegae bv. officinalis

Genomic studies of uncultivated archaea

Abstract | Archaea represent a considerable fraction of the prokaryotic world in marine and terrestrial ecosystems, indicating that organisms from this domain might have a large impact on global energy cycles. However, many novel archaeal lineages that have been detected by molecular phylogenetic approaches have remained elusive because no laboratory-cultivated strains are available. Environmental genomic analyses have recently provided clues about the potential metabolic strategies of several of the uncultivated and abundant archaeal species, including non-thermophilic terrestrial and marine crenarchaeota and methanotrophic euryarchaeota. These initial studies of natural archaeal populations also revealed an unexpected degree of genomic variation that indicates considerable heterogeneity among archaeal strains. Here, we review genomic studies of uncultivated archaea within a framework of the phylogenetic diversity and ecological distribution of this domain

Genomic studies of uncultivated archaea

Abstract | Archaea represent a considerable fraction of the prokaryotic world in marine and terrestrial ecosystems, indicating that organisms from this domain might have a large impact on global energy cycles. However, many novel archaeal lineages that have been detected by molecular phylogenetic approaches have remained elusive because no laboratory-cultivated strains are available. Environmental genomic analyses have recently provided clues about the potential metabolic strategies of several of the uncultivated and abundant archaeal species, including non-thermophilic terrestrial and marine crenarchaeota and methanotrophic euryarchaeota. These initial studies of natural archaeal populations also revealed an unexpected degree of genomic variation that indicates considerable heterogeneity among archaeal strains. Here, we review genomic studies of uncultivated archaea within a framework of the phylogenetic diversity and ecological distribution of this domain

Screening for novel genes of Saccharomyces cerevisiae involved in recombinant antibody production.

Cost-effective manufacturing of biopharmaceuticals in non-mammalian hosts still requires tremendous efforts in strain development. In order to expedite identification of novel leads for strain engineering, we used a transposon-mutagenized yeast genomic DNA library to create a collection of Saccharomyces cerevisiae deletion strains expressing a full-length IgG antibody. Using a high-throughput screening, transformants with either significantly higher or lower IgG expression were selected. The integration site of the transposon in three of the selected strains was located by DNA sequencing. The inserted DNA lay within the VPS30 and TAR1 open reading frame, and upstream of the HEM13 open reading frame. The complete coding sequence of these genes was deleted in the wild-type strain background to confirm the IgG expression phenotypes. Production of recombinant antibody was increased 2-fold in the Δvps30 strain, but only mildly affected secretion levels in the Δtar1 strain. Remarkably, expression of endogenous yeast acid phosphatase was increased 1.7- and 2.4-fold in Δvps30 and Δtar1 strains. The study confirmed the power of genome-wide high-throughput screens for strain development and highlights the importance of using the target molecule during the screening process.Peer reviewe

Enhanced isopropanol-butanol-ethanol (IBE) production in immobilized column reactor using modified Clostridium acetobutylicum DSM792

Peer reviewe

Efficient strategy to alleviate the inhibitory effect of lignin-derived compounds for enhanced butanol production

In the present study, the effect of one of the most important lignin-derived inhibitors (lignosulfonate) was assessed. A technique to overcome the lignosulfonate inhibitory action in the acetone−butanol−ethanol (ABE) fermentation process is proposed here. Different lignosulfonates were primarily added in the fermentation medium to observe their mechanistic action on the ABE production profile. Augmenting lignosulfonate concentration (0.5 g L−1) resulted in a drastically reduced solvent titer (ABE ∼1.50 g L−1). Especially, low-molecular-weight linosulfonate (1 g L−1) severely affected the solvent production and completely ceased the fermentation process. Therefore, a strategic approach that triggers the key genes responsible for butanol production was explored. The experimental analysis revealed that soy meal addition could enhance Clostridium acetobutylicum survival in the presence of lignosulfonates (0.25−3 g L−1). Moreover, soy meal addition also enhanced butanol concentration over 1.5-fold as compared to the control experiment. The ABE production using wood hydrolysate also produced substantial solvent titer (ABE ∼11 g L−1) in the presence of soy meal (5 g L−1). The transcriptional analysis results showed that important genes in clostridial metabolic pathways were upregulated in the presence of soy meal addition during fermentation.Peer reviewe

Reducing agents assisted fed-batch fermentation to enhance ABE yields

Acetone-butanol-ethanol (ABE) fermentation process is a promising bioenergy option amid rising concerns over the environmental impact of fossil fuel usage. However, the commercialization of the ABE process has been marred by challenges of low product yield and titer, thereby non-competitive process economics. Here, we coupled cost competitive reducing agents with a controlled feeding strategy to improve both product titer and yield. Reducing agents promote cofactor dependent butanol production, while fed-batch operation enhances glucose consumption, final ABE titer, and partly mitigates product toxicity. The effects of ascorbic acid, L- cysteine, and dithiothreitol (DTT) on ABE fed-batch production using Clostridium acetobutylicum was investigated in current study. NADH, ATP, extracellular amino acid secretion, and NADH-dependent butanol dehydrogenase (BDH) assays were performed to study the metabolic modifications triggered by reducing agents. Incidentally, L- cysteine and DTT improved ABE solvent titer by 2-fold, producing 24.33 and 22.98 g/L ABE with solvent yields of 0.38 and 0.37 g/g, respectively. Elevated NADH, BDH, and ATP levels in fermentation broth reflected in enhanced ABE titer and yield. Furthermore, histidine secretion emerged as an important factor in Clostridial acid stress tolerance in this study. The results demonstrate that addition of reducing agents in fed-batch ABE fermentation operation enables efficient utilization of glucose with significant improvement in solvent production.Peer reviewe