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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Ecophysiology of novel intestinal butyrate-producing bacteria
    Bui, Thi Phuong Nam - \ 2016
    Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Caroline Plugge. - Wageningen : Wageningen University - ISBN 9789462577015 - 202
    butyrates - butyric acid bacteria - intestines - microbial interactions - faecal examination - mice - man - infants - genomics - intestinal physiology - microbial physiology - biochemical pathways - lysine - sugar - butyraten - boterzuurbacteriën - darmen - microbiële interacties - fecesonderzoek - muizen - mens - zuigelingen - genomica - darmfysiologie - microbiële fysiologie - biochemische omzettingen - lysine - suiker

    The human intestinal tract harbours a trillion on microbial cells, predominantly anaerobes. The activity and physiology of these anaerobes is strongly associated with health and disease. This association has been investigated for a long time.However, this has not been fully understood. One of the reasons is the limited availability of cultured representatives. It is estimated that there may be more than 3000 species colonised in the gut of healthy individuals, however, only a bit over 1000 species have been isolated and characterised. Among the intestinal microbes, butyrate-producing bacteria are of special interest as the butyrate produced, is crucial to maintain a healthy gut. In addition, butyrate-producing bacteria have shown a reverse correlation with several intestinal diseases. In Chapter 2 we described a novel species Anaerostipes rhamnosivorans 1y2T isolated from an infant stool. This strain belonged to genus Anaerostipes within Clostridium cluster XIVa. A. rhamnosivorans had a capability of converting rhamnose into butyrate that is unique within intestinal butyrate-producing bacteria. The genomic analysis also revealed the entire rhamnose fermentation pathway as well as the acetyl-CoA pathway for butyrate production. This bacterium is able to produce butyrate from a wide range of sugars as well as lactate plus acetate. In Chapter 3, we described the microbial interactions between A. rhamnosivorans and Bacteriodes thetaiotaomicron in dietary pectins; Blautia hydrogenotrophica in lactate and small amount of acetate; Methanobrevibacter smithii in glucose. We observed that A. rhamnosivorans was able to benefit from its partners in all cocultures for butyrate production. This is likely due to its high metabolic flexibility. While the interaction between A. rhamnosivorans and B. thetaiotaomicron appeared as syntrophy, the interaction between A. rhamnosivorans and hydrogenotrohic microbes were cross-feeding type where hydrogen was transferred between two species. The latter resulted in an increase in butyrate level. In Chapter 4 we described a novel species Intestinimonas butyriciproducens SRB521T representing a novel genus Intestinimonas from a mouse caecum within Clostridium cluster IV. This bacterium produced butyrate and acetate as end products from Wilkins-Chalgren-Anaerobe broth.

    Butyrate production is assumed to derive from carbohydrate employing acetyl-CoA pathway. No gut bacterium is known to convert proteins or amino acids to butyrate although butyrogenic pathways from amino acid degradation have been detected in the human gut using metagenomic approach. In Chapter 5 we discovered a novel butyrate synthesis pathway from the amino acid lysine and the Amadori product fructoselysine in Intestinimonas butyriciproducens AF211 that was isolated from human stool. This strain appeared to grow much better in lysine as compared to sugars although lysine and acetyl-CoA pathways were both detected in its complete genome. Moreover, the strain AF211 was able to metabolise efficiently fructoselysine into butyrate, and acetate was found to affect the fructoselysine fermentation, representing the impact of the environmental conditions where acetate is abundant in the gut. While the lysine pathway was found in the gut of many individuals, the fructoselysine pathway was present in only half of those samples. The finding that strain I. butyriciproducens AF211 is capable of the butyrogenic conversion of amino acid lysine and fructoselysine, an Amadori product formed in heated foods via the Maillard reaction, indicated a missing link that coupling protein metabolism and butyrate formation. As this Amadori product has been implicated to play a role in aging process, the use of strain AF211 as fructoselysine clearance in the gut needs further investigation. In Chapter 6 we performed genomic and physiological comparison between the I. butyriciproducens strain AF211 (human isolate) and SRB521T (mouse isolate). I. butyriciproducens was the most abundant species within the Intestinimonas genus and highly prevalent in humans based on metadata analysis on 16S amplicons. We confirmed that the butyrogenesis from lysine was a shared characteristic between the two I. butyriciproducens strains. We also observed the host specific features including tolerance to bile, cellular fatty acid composition, more efficient capability of converting sugars into butyrate, especially galactose and arabinose, in the human strain AF211. In addition, genomic rearrangements as well as variations in bacteriophages differed among strains.

    Exploring the ecophysiology of anaerobic communities of methanotrophic archaea and sulfate-reducing bacteria
    Timmers, P.H.A. - \ 2015
    Wageningen University. Promotor(en): Fons Stams, co-promotor(en): Caroline Plugge. - Wageningen : Wageningen University - ISBN 9789462575820 - 181
    sulfate reducing bacteria - methane - oxidation - anaerobic conditions - sulfates - marine sediments - microbial physiology - sulfaat reducerende bacteriën - methaan - oxidatie - anaërobe omstandigheden - sulfaten - mariene sedimenten - microbiële fysiologie

    Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) is a widespread occurring process in anoxic marine sediments. The process is performed by ANaerobic MEthane oxidizing archaea (ANME) and associated sulfate reducing bacteria (SRB). The ANME presumably oxidize methane through reverse methanogenesis. The associated SRB were thought to reduce sulfate using an interspecies electron carrier (IEC) derived from AOM. The product of methane oxidation that is transferred to the SRB is either a less reduced compound that acts as IEC or electrons are transferred directly (through nanowires or pili) or indirectly (through extracellular quinones). However, recent evidence emerged that ANME could perform both methane oxidation and sulfate reduction to produce sulfur, where the SRB disproportionate the produced sulfur. Little is known on the physiology and ecology of these ANME and associated SRB. The main reasons for this are the difficulties in lab cultivation and to perform in situ studies.

    Anaerobic methane oxidation is a process that is at the border of what is energetically possible for sustaining life, which makes it hard to cultivate the responsible organisms. Estimates of the Gibbs free energy yields are between -18 and -35 kJ mol-1 and growth rates between 1.1 and 7.5 months, depending on the environment. AOM therefore operates close to thermodynamic equilibrium and is highly dependent on substrate and product concentrations. In chapter 2, we obtained faster growth rates at elevated methane partial pressure as compared to ambient pressure. The increase in partial pressure increased the solubility of methane and thus the energy yield for the organisms. In chapter 6, we showed higher AOM activity and growth of ANME under thermodynamically favorable sulfate and sulfide concentrations. The problems in studying the process in situ in complex environments comes from difficulties in differentiation of reversible processes. In most studies, methane oxidation is monitored by labelled CO2 formation from labelled methane. Methanogens can perform trace methane oxidation (TMO)during net methanogenesis, which also results in the production of labelled CO2 from labelled methane. When AOM becomes less favorable, the anaerobic back flux of AOM becomes significant, leading to the production of measurable amounts of methane. In chapter 2 and chapter 3, we were able to differentiate between AOM and TMO in long-term incubations.

    Another challenge is related to the detection of ANME in complex environments. The phylogenetic distance between and within ANME clades is large. In chapter 5, we discussed the difficulties in primer and probe design for selective detection of ANME without targeting closely related methanogens. Furthermore, it is not known if even more ANME species and clades have yet to be discovered that are not detected with the primers and probes used thus far. In chapter 3, we found indications that besides ANME-2a/b, ANME-2d archaea were also able to perform AOM coupled to sulfate reduction in freshwater conditions. The finding of ANME-2a/b in freshwater shows that ANME archaea are ubiquitously distributed and not only occur in marine sediments. In chapter 6, we confirmed that different ANME clades show niche separation based on the presence of methane and different sulfate and sulfide concentrations. In chapter 2, we obtained indications that ANME-2c grows at high methane partial pressure. More research on the ecophysiology could help in understanding occurrence and activity of ANME in different environments.

    Many different SRB have been found so far to form close associations with ANME. Most fall within the Desulfosarcina/Desulfococcus (DSS) clade and only for two enrichment cultures the dominant partner of ANME-2a/b was determined to belong to a specific group with the DSS named SEEP-SRB1. In chapter 2, we found more evidence that a group outside the DSS clade, SEEP-SRB2, could also associate with ANME-2a/b and that Eel-1 members are not directly involved in AOM. In chapter 4, we enriched for SRB within the DSS clade on alternative substrates besides methane, but we were unable to show that these are involved in AOM. Therefore, more research on the sulfate-reducing partner is needed to understand the metabolic interactions between ANME and SRB.

    A physiologically based kinetic model for bacterial sulfide oxidation
    Klok, J.B.M. ; Graaff, C.M. de; Bosch, P.L.F. van den; Boelee, N.C. ; Keesman, K.J. ; Janssen, A.J.H. - \ 2013
    Water Research 47 (2013)2. - ISSN 0043-1354 - p. 483 - 492.
    afvalwaterbehandeling - biotechnologie - zwavelwaterstof - oxidatie - ontzwaveling - alkalibacillus haloalkaliphilus - microbiële fysiologie - afvalwaterbehandelingsinstallaties - waste water treatment - biotechnology - hydrogen sulfide - oxidation - desulfurization - alkalibacillus haloalkaliphilus - microbial physiology - waste water treatment plants - sulfur-oxidizing bacteria - biologically produced sulfur - dissolved sodium sulfide - parameter-estimation - hydrogen-sulfide - soda lakes - bioreactors - thiosulfate - mechanisms - pathways
    In the biotechnological process for hydrogen sulfide removal from gas streams, a variety of oxidation products can be formed. Under natron-alkaline conditions, sulfide is oxidized by haloalkaliphilic sulfide oxidizing bacteria via flavocytochrome c oxidoreductase. From previous studies, it was concluded that the oxidation-reduction state of cytochrome c is a direct measure for the bacterial end-product formation. Given this physiological feature, incorporation of the oxidation state of cytochrome c in a mathematical model for the bacterial oxidation kinetics will yield a physiologically based model structure. This paper presents a physiologically based model, describing the dynamic formation of the various end-products in the biodesulfurization process. It consists of three elements: 1) Michaelis–Menten kinetics combined with 2) a cytochrome c driven mechanism describing 3) the rate determining enzymes of the respiratory system of haloalkaliphilic sulfide oxidizing bacteria. The proposed model is successfully validated against independent data obtained from biological respiration tests and bench scale gas-lift reactor experiments. The results demonstrate that the model is a powerful tool to describe product formation for haloalkaliphilic biomass under dynamic conditions. The model predicts a maximum S0 formation of about 98 mol%. A future challenge is the optimization of this bioprocess by improving the dissolved oxygen control strategy and reactor design.
    Ecophysiology of microorganisms in microbial elctrolysis cells
    Croese, E. - \ 2012
    Wageningen University. Promotor(en): Fons Stams; G.J.W. Euverink, co-promotor(en): J.S. Geelhoed. - S.l. : s.n. - ISBN 9789461733047 - 149
    microbiële fysiologie - ecofysiologie - elektrolyse - microbiële brandstofcellen - microbial physiology - ecophysiology - electrolysis - microbial fuel cells

    One of the main challenges for improvement of the microbial electrolysis cell (MEC) has been the reduction of the cost of the cathode catalyst. As catalyst at the cathode, microorganisms offer great possibilities. Previous research has shown the principle possibilities for the biocathode for H2 production with mixed microbial communities. In this thesis we analyzed the microbial communities from several biocathodes for H2 production. The microbial population of the very first MEC biocathode for H2 production (Chapter 2) showed a dominant population of Desulfovibrio spp.. A member of those dominant species, Desulfovibrio strain G11 was reinoculated in a biocathode and produced current and H2. On basis of previous knowledge of known Desulfovibrio spp., the molecular mechanism of electron uptake from a cathode with H2 production was proposed to have similarities to mechanisms that have been proposed for syntrophic growth.
    In Chapter 3 the microbial population of 5 more MEC biocathodes was analyzed. Those MECs were fed with either acetate or bicarbonate and consisted of two different designs. The results showed that the microbial communities from the same setup design are more similar than fed with the same carbon source. Furthermore, ribotypes from the phyla, Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria were found to be dominant. To understand more on the mechanisms of H2 production in the MEC, a hydrogenase gene microarray was used to analyze the hydrogenase genes present in 3 of the cathode samples. Those results showed that genes coding for bidirection NAD(P) dependent hydrogenases were mostly present in the MEC biocathode. Those results suggest a mechanisms involving cytoplasmatic NAD(P) dependent hydrogenases rather than energy converting hydrogenases as proposed before.
    To understand the molecular mechanisms it is important to obtain pure cultures from the MEC biocathode and test them for biocathode activity. In chapter 4 we describe a Citrobacter species strain PS2 which was isolated from the MEC biocathode. PS2 was very similar to other Citrobacter spp. able to produce fermentative H2 from a diversity of carbon sources. When inoculated in the MEC biocathode fed with pyruvate, current increased and H2 was produced with comparable efficiencies and production rates as mixed cultures biocathodes. Addition of membrane potential uncouplers nigericin and monensin showed no change in current and H2 production rates, suggesting that the molecular mechanism does not involve membrane potential driven processes.
    Finally, in chapter 5, we explored the usefulness of statistical methods to pinpoint which species are most important for MEC performance. We analyzed DGGE profiles from 5 different MEC anodes using two distinct statistical techniques, Radundacy analysis (RDA) and QR factorization (QRE), and tried to link those profiles to experimental data current, resistance, potential and overpotential. The results showed that current was mostly related to species composition and we were able to pinpoint a few band from DGGE that were influencing changes in experimental parameters most. The results showed that both RDA and QRE are useful methods, of which RDA takes all bands into account, but is therefore less precise; QRE is numerical precise but by eliminating bands that explain least of the variation and therefore using QRE might neglect effect of those bands. Altogether, RDA with additional QRE is useful to give an indication of which species from a mixed community are most likely important for MEC performance and can be used to find a focus in mixed community analysis.
    From our results we conclude that a large diversity of bacteria is able to catalyze the biocathodes reaction for H2 production. The species that develop at a cathode might be largely influenced by the design of the used setup, which has to be considered when comparing different experiments. In addition, our results suggest that a general mechanism, present in many different bacterial species, is involved in MEC H2 production. We propose a molecular mechanism involving a series of cytochromes and cytoplasmatic H2 production by NAD(P)+ dependent bidirectional hydrogenases that use energy from electrons derived from the cathode. The biocathode is a promising technology for application in the MEC, although to date the chemical cathodes still outcompete the biocathode, the biocathode offers great possibilities for future applications including production of other products such as ethanol, methane, succinate or acetate.

    Fine tuning of the Bacillus cereus stress respons: role of transcriptional regulators
    Voort, M. van der - \ 2008
    Wageningen University. Promotor(en): Tjakko Abee; Willem de Vos. - [S.l.] : S.n. - ISBN 9789085048985 - 143
    bacillus cereus - stressreactie - microbiële fysiologie - genexpressie - transcriptie - transcriptiefactoren - regulator-genen - bacillus cereus - stress response - microbial physiology - gene expression - transcription - transcription factors - regulatory genes
    The bacterium Bacillus cereus is able to survive and grow in a high diversity of environments, including foods, such as milk and pasta. Consequently, B. cereus can cause foodborne illnesses and food spoilage. During its time in food B. cereus encounters many changes in its environment, as the food is for instance heated and eaten. In order to cope with these changes in its environment B. cereus is able to switch on different sets of genes. This so-called gene regulation is regulated by an array of regulators. The performed research expands the understanding of the role of these regulators in fine tuning of gene regulation in response to changing environment in food.
    Virussen : op de grens van leven en dood : bestrijding kan niet; infectie voorkomen is de enige weg
    Heuvelink, E. ; Kierkels, T. - \ 2007
    Onder Glas 4 (2007)3. - p. 10 - 11.
    gewasbescherming - plantenvirussen - Pepinomozaïekvirus - microbiële fysiologie - infecties - fotosynthese - geïntegreerde bestrijding - potplanten - snijbloemen - teelt onder bescherming - bedrijfshygiëne - glastuinbouw - plant protection - plant viruses - pepino mosaic virus - microbial physiology - infections - photosynthesis - integrated control - pot plants - cut flowers - protected cultivation - industrial hygiene - greenhouse horticulture
    Virussen geven in sommige teelten veel problemen. Denk bijvoorbeeld aan het pepimozaïekvirus. Een virus kost de plant energie en bouwstoffen, belemmert de fotosynthese en tast de sierwaarde van snijbloemen en potplanten ernstig aan. Alle reden om te streven naar virusvrije gewassen. Zelfs als er geen uiterlijke symptomen zijn. Een reportage over de groei, bouw en werking van (planten)virussen en gewassen
    Molecular microbial ecology manual
    Kowalchuk, G.A. ; Bruijn, F.J. de; Head, I.M. ; Akkermans, A.D.L. - \ 2004
    Dordrecht : Kluwer - ISBN 9781402021831
    microbiologie - microbiële ecologie - microbiële fysiologie - methodologie - micro-organismen - ecologie - biochemie - microbiology - microbial ecology - microbial physiology - methodology - microorganisms - ecology - biochemistry
    The field of microbial ecology has been revolutionized in the past two decades by the introduction of molecular methods into the toolbox of the microbial ecologist. This molecular arsenal has helped to unveil the enormity of microbial diversity across the breadth of the earth's ecosystems, and has revealed that we are only familiar with a very small minority of the organisms that carry out key microbial functions in diverse habitats. The Molecular Microbial Ecology Manual, Second Edition (MMEM-II) provides a detailed and user-friendly description of the methods that have made this revolution in microbial ecology possible. However, what is perhaps most exciting about MMEM-II is that it contains a large number of new chapters, highlighting the newest trends in microbial ecology research, which seek to provide more quantitative and statistically robust data, and means of coupling microbial identity and function. In addition, the majority of the proven methods described in MMEM's first version have undergone significant revisions to provide the most up-to-date applications available. The state-of-the-art methods described in MMEM-II have not only been provided by experts in the field, but in most cases by the laboratories that actually first developed and applied the methods, thus providing the MMEM-II user with unique first-hand tips and insight.
    Biochemistry and physiology of syntrophic propionate-oxidizing microbial consortia
    Bok, F. de - \ 2002
    Wageningen University. Promotor(en): W.M. de Vos; A.J.M. Stams. - S.l. : S.n. - ISBN 9789058085726 - 118
    propionzuurbacteriën - propionibacteriaceae - biochemie - microbiële fysiologie - propionic acid bacteria - propionibacteriaceae - biochemistry - microbial physiology
    Industrial wastewaters can be purified in methanogenic bioreactors such as the upflow anaerobic sludge bed (UASB) reactor. In such reactors biomass is completely degraded to CH 4 and CO 2 , by several trophic groups of microorganisms. However, those gases are also responsible for the enhanced greenhouse effect, and therefore the occurrence of methanogenic decomposition in natural habitats is much less appreciated.

    In the introduction ( Chapter 1 ) of this thesis, one particular step in the process of methanogenic decomposition, propionate oxidation, is discussed. Propionate is one of the compounds which can only be oxidized syntrophically under methanogenic conditions, and the organisms responsible for this step are extremely interesting due to their ability to thrive at the lowest possible amounts of energy available.

    Syntrophic propionate-oxidizing bacteria produce acetate, H 2 , CO 2 and formate. To make this reaction energetically feasible, methanogenic archaea must keep the concentrations of H 2 and formate extremely low. Two classes of enzymes which may be important for this 'Interspecies electron transfer', i.e. hydrogenase and formate dehydrogenase, and the present knowledge of these enzymes, is discussed in Chapter 1. Furthermore, this Chapter summarizes what is know about syntrophic propionate-oxidizing bacteria so far.

    Most of the isolated propionate-oxidizing bacteria belong to the genus Syntrophobacter and cluster together with gram-negative sulfate reducing bacteria within thed-subdivision of the proteobacteria. Other syntrophic propionate oxidizers are more closely related to the genus Syntrophus , while recently two thermophilic organisms were described which are close relatives of low GC gram-positive Desulfotomaculum species. A related organism was also enriched from mesophilic sludge, and Chapter 2 of this thesis describes the isolation and physiological characterization of a defined co-culture of this organism with Methanospirillum hungatei . Remarkably this organism, which was named ' Pelotomaculum schinkii' , harbors two different 16S rDNA genes. This unusual property of sequence heterogeneities has also been reported for other gram-positive bacteria. However, compared to other syntrophic propionate oxidizers this organism is exceptional since it did not grow axenically on any of the substrates tested. For most syntrophic propionate oxidizing bacteria it is known that propionate is oxidized via the Methyl-malonyl-CoA pathway. Besides the ability to couple propionate oxidation to the sulfate reduction, these organisms are also able to ferment fumarate in pure culture. Although ' Pelotomaculum schinkii' most likely uses the methyl-malonyl-CoA pathway to oxidize propionate, none of the intermediates of this pathway supported growth in this organism. Therefore, this organism may be the first true obligate syntrophic anaerobic bacterium isolated.

    An organism, which does not use the methyl-malonyl-CoA pathway to oxidize propionate, is Smithella propionica . This organism produces small amounts of butyrate from propionate and it produces much less CH 4 than organisms, which are known to use the methyl-malonyl-CoA pathway. The occurrence of another pathway of propionate conversion was previously already demonstrated in methanogenic habitats and enrichment cultures. The randomizing methyl-malonyl-CoA pathway could not account for the products recovered from 13C-labeled propionate in these studies. Therefore the pathway of propionate oxidation was studied with 13C-NMR in Smithella propionica ( Chapter 3 ). Experiments with 2,3- 13C-labeled propionate revealed that half of the methyl-methylene bonds in propionate were broken during propionate conversion. Labeled bicarbonate was never recovered from cultures grown on labeled propionate, and experiments in which 13C-bicarbonate was added showed that it was not incorporated either. These observations were inconsistent with pathways proposed previously to occur in methanogenic habitats, such as the acryloyl-CoA pathway and the reductive carboxylation pathway. In this Chapter, a novel pathway of propionate is proposed. The results of 13C-NMR experiments suggested that the C2 of propionate is coupled to the carboxyl-group of a second propionate molecule, followed by a rearrangement to a 6-carbon unbranched intermediate. Cleavage of this molecule then yields acetate and butyrate, which is further oxidized syntrophically to acetate. Such pathway perfectly explained the ratios of labeled products recovered from experiments with 1-, 2-, 3- and 2,3- 13C-labeled propionate, and also fitted with the stoichiometry of propionate conversion. In batches to which 1- and 2- 13C-labeled acetate was added together with unlabeled propionate, a small amount of label was recovered in propionate revealing that the proposed pathway should be reversible.

    For all other studies described in this thesis Syntrophobacter fumaroxidans was used as a model organism. Previous studies have revealed that this organism is able to produce both H 2 and formate during propionate oxidation, and it could only grow with methanogens that utilize both H 2 and formate. The enzymes responsible for proton and bicarbonate reduction are hydrogenase and formate dehydrogenase. The presence of both enzymes in Syntrophobacter fumaroxidans was also demonstrated in previous studies. In Chapter 4 of this thesis several experiments are described which demonstrate that this organism possesses at least 2 distinct formate dehydrogenases and at least 3 distinct hydrogenases. All these enzymes were induced under all growth conditions tested, though there were some variations in the levels of each enzyme. One of the formate dehydrogenases may be involved in CO 2 -fixation by running the acetyl-CoA cleavage pathway in the reverse direction during growth on propionate. Most likely the other enzyme is involved in terminal reduction of CO 2 .

    The presence of multiple hydrogenases in sulfate-reducing bacteria is not unusual. For the genus Desulfovibrio a model has been described how these organism use three distinct hydrogenases to conserve energy. Such a H 2 -cycling mechanism may also be present in S. fumaroxidans . In a separate study ( Chapter 5 ), the hydrogenase and formate dehydrogenase levels of axenic S. fumaroxidans cells were compared to the levels in cells which were grown syntrophically. Since the methanogenic partner of syntrophic growth ( Methanospirillum hungatei ) also possesses these enzymes, the organisms needed to be separated from each other in order to analyze the levels in S. fumaroxidans . Percoll gradient centrifugation proved to be an excellent method to separate syntrophically grown cells. The S. fumaroxidans cells which were separated in this way exhibited very high formate dehydrogenase activities as compared to cells grown axenically. Also the M. hungatei cells which were grown syntrophically exhibited higher formate dehydrogenase activities as compared to cells grown axenically on H 2 or formate. The hydrogenase levels of these cells were comparable to the levels in axenic cultures. For S. fumaroxidans the hydrogenase levels in cells which were grown syntrophically were also higher than in cells which were grown axenically. Both enzymes seemed to play an important role in S. fumaroxidans during syntrophic growth, while the enzyme levels of M. hungatei suggested a more important role for formate dehydrogenase during syntrophic growth. One of the hydrogenase which could be involved in syntrophic growth was purified from S. fumaroxidans cells grown on fumarate ( Chapter 6 ). The enzyme is a typical NiFe-hydrogenase though the levels of both H 2 -uptake and H 2 -evolution were relatively high compared to other described [NiFe]-hydrogenases. Electron Paramagnetic Resonance (EPR) experiments predicted the presence of both [4Fe-4S]-, and [3Fe-4S] clusters. The hydrogenase encoding DNA region was amplified by using a combination of PCR and inverse PCR and primers based on the N-terminal sequence of the large subunit and a conserved region of NiFe-hydrogenases from other organisms. The sequence harbored binding motifs for two [4Fe-4S] clusters and one [3Fe-4S] cluster, and a twin-arginine motif in the precursor of the small subunit suggesting a periplasmic location of the enzyme. The protein sequences of both subunits of the enzyme showed highest similarity to the enzymes isolated from Desulfovibrio species. Previous studies revealed that in those organisms could catalyze the reduction of protons during syntrophic growth on lactate. Therefore, it is likely that the enzyme isolated from S. fumaroxidans is also used to reduce protons to H 2 during syntrophic growth on propionate.

    The two formate dehydrogenases of S. fumaroxidans were both isolated and appeared to be enzymes with very high specific activities, both in the direction of formate oxidation as well as in the direction of CO 2 -reduction ( Chapter 7 ). Since a function as formate dehydrogenase was very unlikely during growth of this organism, a more appropriate name for these enzymes would be 'CO 2 -reductases'. Both enzymes could be isolated from fumarate grown cells, but also from cells grown syntrophically on propionate. EPR-experiments revealed the presence of [2Fe-2S] clusters in FDH1, while [4Fe-4S] clusters were detected in both enzymes. The presence of tungsten could not be confirmed with EPR spectroscopy, but metal-analysis demonstrated that both these enzymes were tungsten-selenium proteins and that they did not contain molybdenum. From a comparison of these two enzymes with formate dehydrogenases isolated from other organisms, it was concluded that the few formate dehydrogenases described with a CO 2 -reducing function contain tungsten. Enzymes, which physiologically mainly oxidize formate, may contain either W or Mo, but preferably molybdenum since this compound is present at much higher concentrations in natural environments. It is hypothesized in this Chapter that the Mo-containing enzymes evolved from W-containing enzymes.

    In Chapter 8 a new hypothesis is proposed for the origin of the eukaryotic cell. The first eukaryote is proposed to have arisen in a methanogenic ecosystem. The host was a heterotrophic fermentative organism, which produced reduced organic compounds. Two endosymbionts were acquired via endocytosis, a acetogenic syntrophic bacterium, and its partner organism a methanogenic archaeon. All partners involved profited from the new situation, which explains the origin of both the nucleus and the mitochondrion in eukaryotic cells. We believe that, among contemporary syntrophic bacteria, the most plausible candidate for a mitochondrial ancestor is a syntrophic propionate oxidizing bacterium.

    Finally, it can be concluded that syntrophic propionate-oxidizing bacteria possess a unique metabolism and that they may have been involved in the crucial step of evolution. Clearly, these organisms deserve more attention in the future!

    Biochemistry and physiology of halorespiration by Desulfitobacterium dehalogenans
    Pas, B.A. van de - \ 2000
    Agricultural University. Promotor(en): W.M. de Vos; A.J.M. Stams. - S.l. : S.n. - ISBN 9789058083494 - 141
    biodegradatie - biologische behandeling - organische halogeenverbindingen - microbiële fysiologie - biochemie - fysiologie - biodegradation - biological treatment - organic halogen compounds - microbial physiology - biochemistry - physiology

    Halorespiration is a novel respiratory pathway, which has been discovered as a result of the search for microorganisms that can be used in bioremediation of chlorinated compounds. Halorespiring bacteria are able to use these compounds as terminal electron acceptor for growth in anaerobic environments. These bacteria have developed enzyme systems with high dechlorination rates and low threshold values. These characteristics are important for the application of dechlorinating bacteria in bioremediation.

    Figure 1
    Figure 1. A 16S rRNA based phylogenetic tree reflecting the relationships of halorespiring bacteria (marked *) with other bacteria.

    The diversity of bacteria capable of using chlorinated compounds as terminal electron acceptor indicates that halorespiration is widespread throughout the bacterial domain (Fig 1). Insight in the physiology and biochemistry of these bacteria is currently lacking. This study aimed to get a better comprehension of the biochemistry of halorespiration. The research has focused on three topics:

    1. elucidation of the coupling of reductive dechlorination to ATP formation in Desulfitobacterium dehalogenans,
    2. isolation and characterization of dehalogenases from different Desulfitobacterium species, and
    3. isolation and characterization of a novel Desulfitobacterium strain from human feces.

    In Chapter 1 , an overview is given of microbial dehalogenation mechanisms with emphasis on halorespiration. The halorespiring bacteria that have been obtained in pure culture, the current models for 3-chlorobenzoate and tetrachloroethene (PCE) respiration, and the characteristics of reductive dehalogenases, are also reviewed.

    Desulfitobacterium dehalogenans is an anaerobic Gram-positive bacterium that uses ortho -chlorinated phenolic compounds as terminal electron acceptor for growth. In Chapter 2, the growth yields of D. dehalogenans grown with hydrogen, formate, pyruvate, or lactate as electron donor and Cl-OHPA as electron acceptor have been compared. In addition, the activities of the different electron donating and electron accepting enzymes were localized. These results indicate that the oxidation of lactate and pyruvate coupled to the reduction of Cl-OHPA yields 1 ATP per mole of acetate produced by substrate level phosphorylation. When formate or hydrogen is used as electron donor for reductive dechlorination, the growth yield is approximately 1/3 of the growth yield with pyruvate as electron donor. Under these growth conditions, energy cannot be conserved via substrate-level phosphorylation.

    However, a proton motive force (PMF) may be established, which can be used by a proton-driven ATPase for ATP-formation. A model has been postulated in which the localization of the electron-donating enzyme (e.g. hydrogenase, formate dehydrogenase, lactase dehydrogenase, or pyruvate ferredoxin oxidoreductase) determines whether a PMF is established. In contrast to the electron transport by the electron transport chain (ETC) and the reduction of the chlorinated compound by the reductive dehalogenase, which do not contribute to the PMF. We have investigated the composition of the ETC, which is involved in electron transport from formate to Cl-OHPA in cell suspensions and have compared it with the ETC involved in fumarate respiration with formate as electron donor ( Chapter 3 ). Menaquinone, cytochrome c, and b were components that were found to be present in cells grown with formate and either Cl-OHPA or fumarate. We have demonstrated that these components could be reduced by formate and oxidized upon addition of the induced electron acceptor. This suggests that (a part of) the halorespiratory chain is shared with fumarate respiration. However, the ETCs involved in halorespiration and fumarate respiration are not identical. The involvement of cytochrome b in fumarate respiration could be demonstrated while this was not possible for halorespiration. The results suggest that cytochrome b is the direct electron donor for fumarate reductase.

    The electron transport chain from formate to Cl-OHPA has been investigated in more detail by electron paramagnetic resonance spectroscopy. In these experiments, we have shown that molybdenum, iron-sulfur clusters, cobalamin, a high spin heme and an unknown iron-sulfur cluster are components that were reduced by formate and oxidized by Cl-OHPA. This may indicate that the formate dehydrogenase which is active in halorespiration is a molybdenum and iron-sulfur containing formate dehydrogenase. This enzyme donates its electrons either to cytochrome c, or the electrons are transferred to cytochrome b. The electrons may then be transferred to menaquinone which takes 2 protons from the cytoplasm and, depending on the localization of the reductive dehalogenase, the protons are released at the outside or inside of the cell, as is shown in figure 2 model A and B, respectively. In addition, oxidation of cobalamin, a cofactor of chlorophenol reductive dehalogenase, was observed in cell suspensions upon addition of Cl-OHPA. This observation strongly suggests that the dehalogenase, which we have characterized, is involved in in vivo halorespiration.

    Figure 2 Model AFigure 2 Model B
    Figure 2: The electron transport system of D. dehalogenans catalyzing the oxidation of formate coupled to reductive dechlorination of 3-chloro-4-hydroxyphenyl acetate. It shows two tentative models for the generation of a proton gradient based on the localization of the ortho -chlorophenol reductive dehalogenase at the outer (model A) or the inner aspect (model B) of the cytoplasmic membrane.

    The isolation and characterization of a chlorophenol reductive dehalogenase is described in Chapter 4 . This enzyme was purified anaerobically from a Triton X-100 extract of the membrane fraction. The purified enzyme catalyzed the dechlorination of Cl-OHPA with a V max of 28 units/mg protein and a K m of 20 mM. In addition, the purified dehalogenase catalyzed the reductive dehalogenation of several ortho -chlorinated phenols and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (E m = -440 mV), one [3Fe-4S] cluster (E m = 170 mV), and one cobalamin per 48-kDa monomer. The Co + /Co 2+ transition had an E m of -370 mV. The corresponding gene has been isolated, cloned, and sequenced, and revealed the presence of two closely linked genes: (i) cpr A, encoding the o-chlorophenol reductive dehalogenase, (ii) cpr B, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. Moreover, cprA contains a twin-arginine type signal sequence that is processed in the purified enzyme.

    Besides ortho -chlorinated phenols, D. dehalogenans is able to use other electron acceptors. In Chapter 5 , the influence of other electron acceptors on the induction of dechlorinating activity and on the dechlorinating activity in cell suspensions and cell extracts is described. The results indicate that D. dehalogenans does not have a preferred electron acceptor in batch cultures, but it utilizes several electron acceptors simultaneously. This could be relevant for in situ bioremediation techniques because the presence of multiple electron acceptors in polluted sediments is not unusual.

    While D. dehalogenans is able use ortho -chlorinated phenols as terminal electron acceptors for growth, Desulfitobacterium sp. strain PCE1 is able to use both chlorophenols and PCE and Desulfitobacterium frappieri strain TCE1 can use PCE and TCE. We compared the substrate spectrum of the enzymes in cell extracts of these strains grown with Cl-OHPA or PCE as electron acceptors ( Chapter 6 ). The results indicate that strain PCE1 contains separate enzymes for PCE and chlorophenol dechlorination. This was studied in more detail by the isolation of the chlorophenol reductive dehalogenase and the PCE reductive dehalogenase of strain PCE1 and the PCE/TCE reductive dehalogenase from strain TCE1. Based on the N-terminal sequence, size and substrate spectrum, the chlorophenol reductive dehalogenase of strain PCE1 was found to be very similar to the dehalogenase of D. dehalogenans. The PCE/TCE reductive dehalogenase of strain TCE1 has similar characteristics as have been described for PCE reductive dehalogenase of strain PCE-S. The PCE reductive dehalogenase from strain PCE1 was found to be a novel type of reductive dehalogenase. The enzyme catalyzed the reduction of PCE, and had a low activity with TCE. The purified enzyme had a subunit size of 45 kDa on SDS-PAGE. The activity of this enzyme as well as of the chlorophenol reductive dehalogenase of strain PCE1 was found to be inhibited upon addition of the cobalamin inhibitors 1-iodopropane and NO to cell extracts.

    In Chapter 7 , the isolation and characterization of a new strain of Desulfitobacterium frappieri is described. This isolate is the first Desulfitobacterium strain described that is not able to use chlorinated ethenes or phenols as terminal electron acceptor.

    Keywords :Halorespiration, Desulfitobacterium dehalogenans , anaerobic dechlorination, bioremediation, PCE, chlorophenol.

    Physiology of solvent tolerance in Pseudomonas putida S12
    Isken, S. - \ 2000
    Agricultural University. Promotor(en): J.A.M. de Bont. - S.l. : S.n. - ISBN 9789058082251 - 88
    industriële microbiologie - pseudomonas putida - microbiële fysiologie - industrial microbiology - pseudomonas putida - microbial physiology

    Hydrophobic organic solvents, like toluene, are toxic for living organisms. This toxicity is an important drawback in the environmental biotechnology as well as in the application of solvents in the production of fine chemicals by whole-cell biotransformations. The effects of organic solvents on micro-organisms have been studied extensively. It was shown that the toxicity of hydrophobic organic solvents is mainly caused by the ability of such solvents to intercalate and accumulate in biological membranes.

    In the last decade, however, several strains that can survive the presence of toxic organic solvents have been isolated. One of the solvent-tolerant strains is Pseudomonas putida S12, studied in this thesis. This strain can grow in the presence of a second phase of organic solvents that have a log P O/W (logarithm of the partition coefficient between octanol and water) value equal to or higher than 2.3. P. putida S12 is able to survive the presence of solvents because of different adaptation mechanisms. This strain can suppress the effect of organic solvents on the membranes by a cis to trans isomerization of the unsaturated fatty acids of the membrane.

    A further adaptation mechanism is presented in this thesis. It is shown that cells adapted to toluene posses an active export system for toluene. Therefore, P. putida S12 compensates not only the toxic effects of organic solvents on the membrane, but decrease also actively the amount of the toxic solvent in the cell. In the presence of the efflux system the concentration of a solvent in the bacterial membrane can be below the theoretical equilibrium. This active efflux system depends on the proton motive force.

    Since we reported the presence of an active export system for solvents, it has been suggested repeatedly, that this system is connected with the well-described efflux systems for antibiotics. Indeed, the adaptation of P. putida S12 to toluene enhances the resistance of this strain to various chemically and structurally unrelated antibiotics, with different targets in the cell.

    However, we could demonstrate that efflux system for toluene in P. putida S12 does not export antibiotics. This efflux system is specific for solvents like toluene and p -xylene. Therefore, the adaptation to an organic solvent must activate other mechanisms responsible for the resistance towards these antibiotics. Likely, this is connected to a general stress response. This general stress response may also cause the decrease of the cell-envelope permeability discussed in the thesis.

    The broad effect solvents have even on the solvent-tolerant strain P. putida S12 are demonstrated. The presence of toluene reduces the maximum growth yield and increases the maintenance requirement. Interestingly, other solvents had a similar effect as toluene as long as they reached the same concentration in the bacterial membrane. Not the chemical structure but the amount of solvent accumulated in the bacterial membrane determines the effect of a solvent on the cells. Therefore, results obtained with toluene can be extrapolated to other solvents as well.

    Biochemistry and bioenergetics of syntrophic propionate-oxidizing bacteria
    Kuijk, B.L.M. van - \ 1998
    Agricultural University. Promotor(en): W.M. de Vos; A.J.M. Stams. - S.l. : Van Kuijk - ISBN 9789054857884 - 138
    micro-organismen - biochemie - fysiologie - microbiële fysiologie - bio-energetica - microorganisms - biochemistry - physiology - microbial physiology - bioenergetics

    Propionaat is één van de belangrijke tussenproducten die worden gevormd tijdens de anaërobe afbraak van complex organisch materiaal. In methanogene milieus, zoals zoetwatersedimenten en anaërobe bioreactoren, is de afbraak van propionaat tot acetaat kooldioxide en waterstof of formiaat een endergone omzetting onder thermodynamische standaardcondities. Dit betekent dat deze omzetting niet plaats kan vinden, tenzij de gevormde producten worden weggenomen. Het gevolg is dat propionaat wordt omgezet door zogenaamde syntrofe consortia, bestaande uit propionaat-oxiderende acetogene bacteriën en waterstof- of formiaat-consumerende methanogene archaea. Uit het Grieks vertaald betekent syntroof letterlijk "samen (syn) voedend (trophos)".

    Gedetailleerde bestudering van het metabolisme van de propionaat-oxiderende acetogene bacteriën werd tot nu toe gehinderd door de noodzakelijke aanwezigheid van een waterstof- of formiaat-consumerende partner. Enkele jaren geleden echter werd duidelijk k dat syntrofe propionaat-oxiderende bacteriën ook zonder een methanogene partner kunnen groeien. Dankzij de groei van deze bacteriën in reinculturen werd het in dit proefschrift beschreven onderzoek waarbij biochemische en energetische aspecten van syntrofe propionaat-oxiderende bacteriën werden bestudeerd, mogelijk. In hoofdstuk 1 wordt een inleiding gegeven over syntrofie met speciale aandacht voor de syntrofe omzetting van propionaat. Tevens worden in hoofdstuk 1 eigenschappen gepresenteerd van de enzymen fumaraat reductase/ succinaat dehydrogenase, fumarase en malaat dehydrogenase, omdat deze enzymen werden gezuiverd uit de syntrofe propionaat-oxiderende bacterie stam MPOB, die als modelorganisme werd gebruikt tijdens dit onderzoek. Stam MPOB werd enkele jaren geleden geïsoleerd uit korrelslib van een anaërobe reactor waarmee afvalwater van een suikerfabriek werd gezuiverd. De bacterie is strikt anaëroob; dit betekent dat stam MPOB in aanwezigheid van zuurstof niet kan overleven. Stam MPOB werd geïsoleerd als een propionaat-omzettende syntrofe culture met methanogene archaea, maar kon in reinculture worden verkregen met behulp van het substraat fumaraat.

    Tijdens de uitvoering van dit onderzoek werd bekend dat andere syntrofe propionaat- oxiderende bacteriën, namelijkSyntrophobacter woliniien S. pfennigii,de oxidatie van propionaat kunnen koppelen aan sulfaatreductie in reinculturen. In
    hoofdstuk 2 wordt beschreven dat stam MPOB ook in staat is om sulfaat te reduceren. De groeisnelheid van stam MPOB met propionaat en sulfaat was, net als die van de twee Syntrophobacter soorten, erg laag vergeleken met de groeisnelheden van propionaat- oxiderende sulfaat-reducerende Desulfobulbus soorten.

    Tijdens de oxidatie van propionaat komen bij drie tussenreacties reductieequivalenten vrij, namelijk bij de oxidatie van succinaat tot fumaraat, malaat tot oxaalacetaat, en pyruvaat tot acetyl-CoA. Deze reductie-equivalenten komen vrij in de vorm van waterstof of formiaat, welke vervolgens verwijderd moeten worden door methanogene archaea om de propionaat- oxidatie te kunnen laten voortgaan. Om de oxidatie van succinaat tot fumaraat te vergemakkelijken is het niet alleen van belang dat de waterstof of formiaat concentratie laag gehouden wordt maar ook de fumaraat concentratie. Hiervoor heeft de bacterie een fumarase nodig dat de omzetting van fumaraat tot malaat efficiënt katalyseert. De zuivering van dit enzym uit stam MPOB wordt beschreven in hoofdstuk 3. Het fumarase van stam MPOB bleek instabiel te zijn in aanwezigheid van zuurstof en werd daarom gezuiverd onder anaërobe condities in een anaërobe tent. Het enzym bestaat uit twee subunits die elk een moleculaire massa van 60 kDa hebben. De N-terminale aminozuurvolgorde van het fumarase van stam MPOB vertoonde duidelijke overeenkomsten met onder andere die van twee fumarases uit Escherichia coli. Met behulp van elektron paramagnetische resonantie (EPR) spectroscopie werd de aanwezigheid van een [4Fe-4S] cluster in het fumarase van stam MPOB aangetoond. Dit cluster reageert met het substraat fumaraat. De katalytische eigenschappen van het fumarase lieten zien dat het enzym efficiënter fumaraat omzet tot malaat dan andersom. Dit is belangrijk voor de vergemakkelijking van de energetisch ongunstige succinaat-oxidatie tijdens de omzetting van propionaat.

    In hoofdstuk 4 wordt de zuivering en karakterisering beschreven van het malaat dehydrogenase van stam MPOB. Dit enzym katalyseert één van de tussenreacties tijdens de syntrofe propionaat-oxidatie waarbij reductie-equivalenten vrijkomen, namelijk de NAD- afhankelijke omzetting van malaat naar oxaalacetaat. Het malaat dehydrogenase van stam MPOB bestaat uit twee subunits met een moleculaire massa van elk 35 kDa. De N-terminale aminozuurvolgorde van het enzym bevatte het geconserveerde gebied dat in de meeste malaat dehydrogenases, van zowel planten, dieren als micro-organismen, voorkomt. De affiniteit van het enzym voor oxaalacetaat en NADH was hoger dan de affiniteit voor malaat en NAD, terwijl het enzym in de cel de omzetting van malaat tot oxaalacetaat katalyseert. Dit is mogelijk de reden voor de bijzonder hoge specifieke activiteit van het gezuiverde malaat dehydrogenase van stam MPOB (1728 U/mg eiwit).

    De zuivering van het fumaraat reductase uit stam MPOB, beschreven in hoofdstuk 5, verliep erg moeizaam, hetgeen voornamelijk werd veroorzaakt door de zuurstofgevoeligheid van het enzym en het feit dat het een membraan-gebonden enzym betrof. Het gezuiverde enzym bevatte drie subunits die qua moleculaire massa, namelijk 70,5, 33,5 en 23,5 kDa, overeenkwamen met reeds bekende fumaraat reductases en succinaat dehydrogenases. Ook op grond van de N-terminale aminozuurvolgorde van de twee grootste subunits kon gezegd worden dat het fumaraat reductase van stam MPOB verwant is aan fumaraat reductases en succinaat dehydrogenases van andere micro-organismen. De meeste andere bekende fumaraat reductases en succinaat dehydrogenases bevatten drie verschillende ijzer-zwavel clusters, namelijk een [2Fe-2S], [4Fe-4S] en [3Fe-4S] cluster. Met behulp van EPR- spectroscopie konden deze drie clusters worden aangetoond in membranen van stam MPOB. In het gezuiverde fumaraat reductase konden echter alleen het [2Fe-2S] cluster en het [4Fe-4S] cluster worden aangetoond. Het signaal van het [4Fe-4S] cluster was bovendien erg zwak. Mogelijkerwijs zijn de [3Fe-4S] en [4Fe-4S] clusters gedeeltelijk verloren gegaan tijdens de zuiveringsprocedure, of na blootstelling aan zuurstof gedeeltelijk afgebroken.

    De oxidatie van succinaat tot fumaraat en waterstof of formiaat is verreweg de energetisch ongunstigste tussenreactie tijdens syntrofe propionaat-oxidatie. Omdat deze reactie zelfs bij de voor methanogene archaea laagst haalbare waterstof- of formiaat- coneentraties nog steeds endergoon is, wordt verondersteld dat de bacterie energie moet investeren in de oxidatie van succinaat. Men denkt dat de elektronen die bij de oxidatie van succinaat vrijkomen, via een omgekeerd elektronentransport naar een lagere redox potentiaal moeten worden geleid waarbij de elektronen wel gekoppeld kunnen worden aan de reductie van protonen of bicarbonaat. Het omgekeerd elektronentransport zou worden gedreven door de hydrolyse van2/3ATP.
    Uit groeistudies met stam MPOB, gekweekt op fumaraat met waterstof of formiaat als elektronendonor (hoofdstuk 6), is gebleken dat de bacterie ongeveer 2/3ATP verkrijgt uit de reductie van 1 mol fumaraat. Deze waarde ondersteunt de hypothese dat de bacterie in de omgekeerde reactie 2/3 ATP moet investeren. In hoofdstuk 6 wordt tevens de lokalisering beschreven van enkele enzymen die betrokken zijn bij de reductie van fumaraat De enzymen fumaraat reductase, succinaat dehydrogenase en ATPase zijn gebonden aan de cytoplasmatische membraan. Hydrogenase en formiaat. dehydrogenase zijn slechts zwak geassocieerd met deze membraan, waarschijnlijk aan de periplasmatische zijde. Daarnaast wordt in hoofdstuk 6 beschreven dat stam MPOB twee typen menaquinonen bevat, menaquinon-6 en -7, en twee typen cytochromen, namelijk b-type cytochromen in de membraanfractie en c -type cytochromen in de oplosbare celfractie Er zijn aanwijzingen gevonden dat cytochroom b en menaquinon onderdeel zijn van de elektronentransportketen tijdens de reductie van fumaraat Indien de oxidatie van succinaat in stam MPOB gekoppeld is aan een omgekeerd elektronentransportmechanisme is het waarschijnlijk dat hierbij de elektronen-transportketen die betrokken is bij de reductie van fumaraat, in omgekeerde richting wordt gebruikt. Helaas konden hier tot dusver alleen enkele indirecte aanwijzingen voor worden verkregen. Omdat het niet is gelukt om van stam MPOB actieve membraanvesikels te maken, was het niet mogelijk om het effect van ionoforen en ATPase-remmers te bestuderen op de waterstofproductie uit de omzetting van succinaat tot fumaraat. Alleen met dergelijke experimenten is het mogelijk om te bewijzen dat de succinaat-oxidatie tijdens syntrofe propionaat-oxidatie wordt gedreven door een energieverbruikend omgekeerd elektronentransport.

    Hoofdstuk 7 beschrijft de fysiologische karakterisering van stam MPOB. Stam MPOB wordt gezien als een nieuwe soort van het geslacht Syntrophobacter. Voor stam MPOB wordt de naam Syntrophobacter fumaroxidans voorgesteld, omdat de bacterie in reinculture kon worden verkregen dankzij zijn groei met fumaraat

    Tenslotte worden in hoofdstuk 8 de voorafgaande hoofdstukken samengevat en bediscussieerd.

    The isoelectric point of bacteria as an indicator for the presence of cell surface polymers that inhibit adhesion.
    Rijnaarts, H.H.M. ; Norde, W. ; Lyklema, J. ; Zehnder, A.J.B. - \ 1995
    Colloids and Surfaces. B: Biointerfaces 4 (1995). - ISSN 0927-7765 - p. 191 - 197.
    micro-organismen - biochemie - fysiologie - microbiële fysiologie - microbiële afbraak - chemie - colloïden - adsorptie - oppervlakten - oppervlaktechemie - microorganisms - biochemistry - physiology - microbial physiology - microbial degradation - chemistry - colloids - adsorption - surfaces - surface chemistry
    Physiology of syntrophic propionate oxidizing bacteria
    Houwen, F.P. - \ 1990
    Agricultural University. Promotor(en): A.J.B. Zehnder; A.J.M. Stams. - S.l. : Houwen - 135
    microbiële afbraak - propionzuur - micro-organismen - biochemie - fysiologie - microbiële fysiologie - microbial degradation - propionic acid - microorganisms - biochemistry - physiology - microbial physiology

    Under methanogenic conditions, with protons and carbon dioxide as intermediate and ultimate electron acceptors, complex organic material is degraded in several steps to methane and carbon dioxide. About 15% of the total carbon compounds are degraded via propionate as an intermediate. Propionate is oxidized to acetate, carbon dioxide and hydrogen. For thermodynamical reasons, this reaction can only proceed if the partial pressure of hydrogen is kept very low. Hydrogenotrophic organisms, e.g. methane bacteria or sulphate reducing bacteria, are syntrophic partner in this process of interspecies hydrogen transfer. Recently, however, it was hypothesized that also formate could be an important compound via which the electrons are transferred to the partner organism.

    The aim of this study was to obtain better fundamental understanding of biochemical and physiological aspects of obligate syntrophic propionate oxidizing bacteria. The presence of an obligate partner organism makes such studies very difficult. In this thesis different methods were used to overcome these difficulties: 1) techniques which do not require pure cultures, e.g. the use of specifically labelled compounds, 2) growth of the acetogen in pure culture by either using artificial electron acceptors or metabolic intermediates, and 3) determining the acetogen specific enzymes by subtracting the activities measured for the pure culture of the electron scavenging partner organism from the activities found in a defined biculture.

    Using invivo high-resolution 13C-NMR, evidence was found for the involvement of the succinate pathway in propionate oxidation by a methanogenic coculture (Chapter 1). The addition of [3-13C]-labelled propionate clearly showed succinate as an intermediate, and the ultimate breakdown product acetate was labelled equally in the C-1 and C-2 positions. In addition, denovo synthesis of propionate from propionate was observed. The 13C-label randomized completely between the C-3 and C-2 of propionate. Apparently propionate and succinate were interconverted at a high rate. These results were in accordance with the data published by others.

    The interconversion of propionate and succinate offered the possibility to study the role of carboxylation reactions in propionate metabolism in some anaerobic bacteria (Chapter 3). This was done in a very easy way by the inclusion of [3- 13C] propionate and H 13C0 3 -, which gave insight into the process of randomization and the types of (de)carboxylating enzymes involved. Both the propionate oxidizer in a methanogenic coculture and Syntrophobacterwolinii were shown to degrade propionate via the. succinate pathway involving a transcarboxylase.

    Chapter 4 deals with a two-liquid-phase electron removal system including the artificial, water soluble redox mediator propylviologen sulphonate (PVS). The organic phase dibutylphtalate was used as reservoir for the electron acceptor 2-anilino-1,4-naphtoquinone. In the abiotic two-liquid-phase system, electrons were transfered from the medium into the organic phase. The indicator organism Acidaminobacterhydrogenoformans oxidized glutamate to acetate without evolution of hydrogen (or formate). However, results indicated that the hydrogen partial pressure obtained by this method, was not low enough to clearly influence the metabolism of the bacterium. Besides possible toxicity problems, the relatively low midpoint redox potential of PVS (-390 mV) may have been the problem for efficient electron transfer at the required hydrogen partial pressure of 10-5 atm. to cause a shift in electron flow during glutamate oxidation. In a syntrophic propionate oxidizing coculture the electron scavenging methane bacteria could not be replaced by the artificial electron acceptor. PVS was tested both as redox mediator in the two-liquid-phase system, and as terminal electron acceptor.

    The metabolic intermediates pyruvate and fumarate were tested for growth in pure culture of the propionate oxidizing organism in a methanogenic coculture (Chapter 5). A propionate fermentation was performed with pyruvate as the substrate. 13C-NMR showed the involvement of the succinate pathway in the formation of propionate. The isolated organism, however, did not oxidize propionate in coculture with hydrogenotrophic methanogens. Moreover, a sulphate reducer appeared to be present in the original coculture. A syntrophic sulphidogenic propionate oxidizing coculture was obtained by repeated transfer of the coculture in medium with propionate and sulphate. To test whether the (slow growing) obligate syntrophic acetogen can be grown on other substrates than propionate, could not be tested because of the contaminating organisms.

    Chapter 6 is the first report on enzyme measurements in syntrophic propionate oxidation. As Syntrophobacterwolinii grows in a defined biculture with a Desulfovibrio species, it was possible to use cell-free extracts of a pure culture of the latter organism as a blanc. Most enzymes involved in the succinate pathway, including the key enzyme propionyl-CoA:oxaloacetate transcarboxylase, were demonstrated in S.wolinii . This confirms the results found by 13C-NMR (Chapter 3).

    Further, S.wolinii appeared to have a lower growth yield than Desulfobulbuspropionicus . This difference is explained in terms of energy conservation mechanisms. Comparison of growth rates of three syntrophic propionate oxidizing cocultures showed that hydrogenotrophic sulphate reducers are more efficient than methanogens during interspecies hydrogen transfer. The more negative Gibbs free energy change under sulphidogenic conditions compared to methanogenic conditions, is thought to contribute to this phenomenon.

    The final chapter (7) deals with the use of 13C-NMR in a complex biological system. Propionate degradation was followed in mesophilic methanogenic granular sludge at 55°C. Because of the non-steady conditions, transient intermediary products accumulated in the medium. The addition of fumarate as secondary substrate stimulated propionate conversion. Propionate and succinate appeared to be direct precursors of each other during propionate metabolism. Selective labelling of one of the substrates offered the possibility to study turnovers ofdifferent compounds. Moreover, interrelated biochemical processes could, in this way, be investigated in a relatively easy way.

    Structure-function relationship of flavoproteins : with special reference to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens
    Berkel, W.J.H. van - \ 1989
    Agricultural University. Promotor(en): F. Mueller. - S.l. : Van Berkel - 133
    flavonoïden - steroïden - porfyrinen - chlorofyl - micro-organismen - biochemie - fysiologie - microbiële fysiologie - enzymen - structuuractiviteitsrelaties - flavonoids - steroids - porphyrins - chlorophyll - microorganisms - biochemistry - physiology - microbial physiology - enzymes - structure activity relationships
    In this thesis different studies probing the structurefunction relationship of some flavoproteins are dealt with. The attention has been focused on two central themes:
    The first part of the thesis deals with studies concerning the application of affinity chromatography in order to allow the large scale preparation of apo flavoproteins.
    In the second part of the thesis, different studies are presented concerning the biophysical properties of p-hydroxybenzoate hydroxylase from Pseudomonasfluorescens .
    Conventional methods for the preparation of apo flavoproteins and general properties of PAD-dependent external monooxygenases are reviewed in Chapter 1 . Special attention has been paid to the different studies performed with p-hydroxybenzoate hydroxylase from P.fluorescens .

    Conventional methods to prepare the apoenzyme of p-hydroxybenzoate hydroxylase from P.fluorescens yield relatively low amounts of apoenzyme showing both variable residual and reconstitutable activity. In Chapter 2 a new method is described to overcome this problem.
    Large amounts of stable apoprotein, showing almost no residual activity, have been obtained by use of DTNB-Sepharose covalent affinity chromatography.
    The enzyme can be reconstituted on the column or the dimeric apoprotein can be isolated in the free state. The degree of reconstitution of almost completely recovered enzyme is better than 95% of the original activity.
    The affinity of p-hydroxybenzoate for the apoprotein is comparable to native holoenzyme. The substrate protects the apoprotein from inactivation.
    The apoenzyme also forms a complex with NADPH. The dissociation constant of this complex is even lower than that of the holoenzyme and is strongly dependent on pH and ionic strength of the solution.
    Kinetic experiments show that the enzyme is reconstituted in a fast process, FAD being tightly bound by the apoprotein.

    In Chapter 3 a new and more general applicable method for the large scale preparation of apo flavoproteins is described. Two classes of flavoproteins have been selected to demonstrate the usefulness of the applied hydrophobic interaction chromatography method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained.
    The holoenzyme of lipoamide dehydrogenase from Azotobactervinelandii can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. The biophysical properties of completely recovered reconstituted lipoamide dehydrogenase compare favorable with the properties of native holoenzyme.
    The holoenzyme of butyryl-CoA dehydrogenase from Megasphaera elsdenii cannot be reconstituted when the apoenzyme is bound to the column. However, this is the first report where stable apoprotein can be isolated in the free state. The yield of apoprotein Is more than 50% of starting material. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation.
    At pH 7.0 apo butyryl-CoA dehydrogenase Is in equilibrium between dimeric and tetrameric forms and reassociates to a nativelike tetrameric structure in the presence of FAD.
    Fluorescence-polarization experiments show that the pH- dependent stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands. Unliganded reconstituted enzyme is easily regreened in the presence of a mixture of coenzyme A and sodium sulfide.

    In Chapter 4 the large scale purification of p-hydroxybenzoate hydroxylase from P.fluorescens is described. The highly purified enzyme can be separated into at least five fractions by anionexchange chromatography. All enzyme molecules exhibit the same specific activity and exist mainly in the dimeric form in solution. The observed microheterogeneity of the enzyme can be explained by the (partial) oxidation of Cys-116 in the sequence of the enzyme.
    The separation of the different enzymic forms has allowed the development of a kinetic FPLC method to describe the dissociation behaviour of the dimeric enzyme.
    By chemical modification studies using maleimide derivatives, DTNB and H 2 O 2 , it is shown that sulfenic, sulfinic and sulfonic acid derivatives of Cys-116 are the main products of oxidation.

    In Chapter 5 the chemical modification of cysteine residues in p-hydroxybenzoate hydroxylase from P.fluorescens by several reagents is described. Differential labeling and sequencing radioactive labeled tryptic peptides have allowed the assignment of different cysteine residues involved in enzyme modification.
    Cys-116 Is found to react rapidly and specifically with N-ethylmaleimide without inactivation of the enzyme.
    The enzyme is easily inactivated by mercurial reagents. Enzyme activity can be fully restored upon addition of dithiothreitol. p-Hydroxybenzoate and also the mercurial compounds themselves inhibit the inactivation reaction.
    A spinlabeled derivative of p-chloromercuribenzoate reacts fairly specifically with Cys-152 in N-ethylmaleimide prelabeled enzyme. Modification of Cys-152 decreases drastically the affinity of the enzyme for the substrate p-hydroxybenzoate. The modified enzyme exhibits a somewhat higher affinity for NADPH than the native enzyme.
    Modification by p-chloromercuribenzoate leads to absorption difference spectra showing pH-dependent maxima at 290 and 360 nm. The observed pKa value of about 7.6 is tentatively ascribed to at least one of the three tyrosine residues located in the substrate binding site.
    From the three-dimensional structure of the enzyme-p-hydroxybenzoate complex it can be deduced that Cys-152 is far away from the active site. The modification results strongly indicate that the substrate binding site and Cys-152 are interdependent.

    Both group-specific chemical modification studies and crystallization experiments have not (yet) led to the elucidation of the NADPH binding site of p-hydroxybenzoate hydroxylase from P.fluorescens. In Chapter 6 the NADPH binding site has been probed using the affinity label p-(fluorosulfonylbenzoyl) adenosine.
    The enzyme is slowly inactivated by the reagent in the presence of 20% dimethylsulfoxide. The inactivation, strongly inhibited by NADPH and 2',5' ADP, can be related to the modification of one amino acid residue.
    Steady state kinetics and 2',5' ADP Sepharose affinity chromatography of modified enzyme suggest that the essential residue is not directly involved in NADPH binding.
    From sequencing radioactive labeled peptides It is shown that Tyr-38 Is the main residue protected from modification in the presence of NADPH.
    The refined crystal structure of the enzyme-p-hydroxybenzoate complex at 0.19 nm resolution shows that Tyr-38 is far away from the active site. From model-building studies using computer graphics a potential mode of binding of both NADPH and 5'-(p-sulfonylbenzoyl)adenosine is presented.

    Chemical modification studies ( Chapter 1.8 and 5 ) have Indicated the presence of an ionized tyrosine residue in the vicinity of the flavin prosthetic group of p-hydroxybenzoate hydroxylase from P.fluorescens .
    In Chapter 7 therefore, the pH-dependent spectral properties of free oxidized enzyme in the absence or presence of substrate (analogues) have been studied by various spectroscopic techniques.
    The observed pH-dependent transitions are explained by (de)protonation of a tyrosine residue involved in binding of the hydroxyl group of the substrate. From the crystal structure it is deduced that Tyr-201 is the most likely candidate showing this low pKa value.
    The exact mechanism of the FAD-dependent aromatic hydroxylation reaction is still unclear. The possible role of ionization of Tyr-201 during catalysis is discussed.

    Enkele artikelen over de invloed van lood op micro - organismen in de bodem
    Anonymous, - \ 1976
    Wageningen : [s.n.] (Literatuurlijst / Centrum voor landbouwpublikaties en landbouwdocumentatie no. 3912)
    algen - bibliografieën - biochemie - chemie - indicatorplanten - lood - microbiële flora - microbiële fysiologie - microbiologie - micro-organismen - fysiologie - bodemflora - toxische stoffen - chemische factoren - algae - bibliographies - biochemistry - chemistry - indicator plants - lead - microbial flora - microbial physiology - microbiology - microorganisms - physiology - soil flora - toxic substances - chemical factors
    Micro - organismen als afvalwater - (en andere milieuvervuilings) indicatoren
    Anonymous, - \ 1975
    Wageningen : [s.n.] (Literatuurlijst / Centrum voor landbouwpublikaties en landbouwdocumentatie no. 3765)
    waterorganismen - bibliografieën - biochemie - samenstelling - microbiële fysiologie - micro-organismen - parasitologie - fysiologie - verontreiniging - eigenschappen - rioolwater - afvalwater - aquatic organisms - bibliographies - biochemistry - composition - microbial physiology - microorganisms - parasitology - physiology - pollution - properties - sewage - waste water
    Reductie en conversie van onverzadigde vetzuren door bacterien
    Anonymous, - \ 1974
    Wageningen : [s.n.] (Literatuurlijst / Centrum voor landbouwpublikaties en landbouwdocumentatie no. 3673)
    bibliografieën - biochemie - onverzadigde vetzuren - metabolisme - microbiële fysiologie - bibliographies - biochemistry - unsaturated fatty acids - metabolism - microbial physiology
    Occurrence and properties of bacterial pectate lyases
    Rombouts, F.M. - \ 1972
    Landbouwhogeschool Wageningen. Promotor(en): W. Pilnik. - Wageningen : Pudoc - ISBN 9789022004128 - 132
    biochemie - plantkunde - enzymen - enzymologie - fermentatie - fruitgewassen - lyasen - microbiële fysiologie - micro-organismen - fysiologie - groenten - biochemistry - botany - enzymes - enzymology - fermentation - fruit crops - lyases - microbial physiology - microorganisms - physiology - vegetables

    Some 100 pectolytic bacteria belonging to different genera and species, were obtained by isolation from vegetables and by screening of culture collections. The crude enzyme preparations of 19 of these strains were typed by mutual comparison. Differences in the composition of five commercial fungal 'pectinase' preparations were also studied. Purified endo pectate lyase of Arthrobacter which was studied in detail, appeared to attack pectate far 'less randomly', than endo pectate lyases of Bacillus polymyxa or Pseudomonas. The best substrates for pectate lyases were not pectates but 21 to 44% esterified pectins. A new method for the determination of the number average degree of polymerization of pectic substances was introduced. The literature on pectolytic enzymes was reviewed.

    Influence of moisture and nutrient content of forage plants on fermentation processes
    Wieringa, G.W. - \ 1970
    Wageningen : [s.n.] (Publikatie / Instituut voor bewaring en verwerking van landbouwprodukten no. 219) - 5
    biochemie - groenvoeders - microbiële fysiologie - micro-organismen - fysiologie - plantaardige producten - kuilvoerbereiding - behandeling - biochemistry - green fodders - microbial physiology - microorganisms - physiology - plant products - silage making - treatment
    bioassay to characterize strains and preparations of Bacillus thuringiensis Berliner; Exudates of germinating spores of Bacillus thuringiensis
    Maas Geesteranus, H.P. ; Noordink, J.P.W. ; Anker, C.A. van den - \ 1967
    Wageningen : [s.n.] (Mededeling / Instituut voor plantenziektenkundig onderzoek, Wageningen no. 450,451) - 6
    bacillus - microbiologie - methodologie - technieken - micro-organismen - biochemie - fysiologie - microbiële fysiologie - bacillus - microbiology - methodology - techniques - microorganisms - biochemistry - physiology - microbial physiology
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