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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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    Burkholderia community structure in soils under different agricultural management
    Salles, J.F. - \ 2005
    Leiden University. Promotor(en): J.A. van Veen; J.D. van Elsas, co-promotor(en): J. Balandreau; E. van der Meijden; E.J.J. Lugtenberg. - Wageningen : Ponsen & Looijen BV - ISBN 9789064648861 - 143
    burkholderia - bacteriën - bodembacteriën - bodembiologie - biogeografie - bedrijfsvoering - landbouw - organismen ingezet bij biologische bestrijding - burkholderia - bacteria - soil bacteria - soil biology - biogeography - management - agriculture - biological control agents
    Bacteriën in dekaarde - Een blackbox die geopend kan worden met behulp van moleculaire technieken?
    Baar, J. ; Konings, M.C.J.M. - \ 2005
    Horst : PPO Paddestoelen (Rapport PPO 2005-4) - 16
    bodembacteriën - groeimedia - moleculaire genetica - identificatie - soil bacteria - growing media - molecular genetics - identification
    Electron donor and acceptor utilization by halorespiring bacteria
    Luijten, M.L.G.C. - \ 2004
    Wageningen University. Promotor(en): Willem de Vos; Fons Stams, co-promotor(en): Gosse Schraa. - [S.l.] : S.n. - ISBN 9789085040675 - 132
    anaërobe micro-organismen - anaërobe omstandigheden - bodembacteriën - tetrachloorethyleen - biodegradatie - microbiële afbraak - anaerobes - anaerobic conditions - soil bacteria - tetrachloroethylene - biodegradation - microbial degradation

    Chlorinated ethenes are widespread soil and groundwater pollutants. Over the last 2 decades a lot of effort has been made to understand the degradation mechanisms for these pollutants. In the early eighties reduction of tetrachloroethene (PCE) was observed in anaerobic soil samples, which was shown to be mediated by microorganisms. The first microorganism able to couple the anaerobic reduction of PCE to growth in a process called halorespiration (alternative terms are chlororespiration, chloridogenesis or dehalorespiration) was isolated in 1993. Since then, about 15 bacteria able to reduce PCE metabolically have been isolated. This thesis describes research on different aspects influencing the reductive dechlorination of chlorinated ethenes by anaerobic halorespiring bacteria.

    A new halorespiring bacterium is described in chapter 1. This bacterium, Sulfurospirillum halorespirans PCE-M2, was isolated from a polluted soil near Rotterdam harbor. Strain PCE-M2 is a metabolically versatile bacterium able to use a variety of electron acceptors and electron donors. This new strain is closely related to Dehalospirillum multivorans, but more detailed studies indicated that strain PCE-M2 belongs to the genus Sulfurospirillum, It also appeared that Dehalospirillum multivorans had to be included in this genus. Consequently, it was reclassified to Sulfurospirillum multivorans.

    Members of the genus Sulfurospirillum were originally known for their sulphur, selenate and arsenate respiring properties. Therefore, we screened a number of halorespiring and related bacteria for their metal reducing properties (Chapter 2). It was shown that the reduction of metals such as ferric iron, manganese, selenate and arsenate is a common property amongst halorespiring bacteria. We also investigated the quinone reducing and oxidizing abilities. AU tested bacteria are able to reduce AQDS7 a quinone-bearing humic acid analogue. Some of the tested bacteria (Desulfitobacterium hafniense DP7, Sulfurospirillum barnesii, S. deleyianum and S. arsenophilum) are also able to oxidize AEbQDS coupled to nitrate reduction.

    The influence of some alternative electron acceptors on the reductive dechlorination is discussed in chapter 3. Sulfurospirillum halorespirans preferably reduces nitrate (to ammonium) and then PCE. In contrast, Sulfurospirillum multivorans reduces nitrate only to nitrite, and PCE reduction is blocked irreversibly in the presence of nitrate. In Desulfitobacterium frappieri TCEl, PCE and nitrate are reduced simultaneously in excess of electron donor. Under electron donor limitation PCE reduction was inhibited (Gerritse Bt al., AppI. Environ. Microbiol. 1999, 65, 5212-5221). The influence of nitrate on the reduction of chlorinated ethenes by halorespiring bacteria differs between species and may also depend on the availability of electron donor. Sulphate, which is not used as electron acceptor by chlorinated ethenes respiring bacteria is often found at polluted sites. We have tested the influence of sulphate on halorespiring bacteria (Chapter 3). It appeared that sulphate does not influence these microorganisms. Sulphite however, a possible electron acceptor for Desulfitobacterium species, inhibits the reduction of PCE. This inhibition may be the result of a chemical interaction between sulphite and cobalamine containing dehalogenases. We also studied the adaptation of Sulfurospirillum halorespirans PCE-M2 to different alternative electron acceptors (Chapter 3). Both nitrate and arsenate are reduced by cells pre-grown on PCE, nitrate, arsenate and selenate. This indicates that the enzymes responsible for the reduction of nitrate and arsenate are constitutiveiy present in S, halorespirans. In contrast, PCE and selenate are only reduced by cells pre-grown on PCE or selenate respectively.

    Halorespiring bacteria have a high affinity for hydrogen (H2). H2 may even be the most important electron donor for these organisms in natural environments. We have studied H2^thresh0ld concentrations in pure cultures of halorespiring bacteria (Chapter 4). H2-threshold values between 0.05 and 0.08 nM under PCE-reducing and nitrate-reducing conditions were measured. Furthermore, we measured H2 concentrations at a field site polluted with chlorinated ethenes. PCE and trichloroethene (TCE) reduction can occur at H2 concentrations below 1 nM. However, for the reduction of lower chlorinated ethenes a higher H2 concentration seems to be required.

    Accumulation of cis-l,2-dichloroethene (cis-l,2-DCE) and vinyl chloride (VC) under anaerobic conditions is often observed. The enrichment of two cultures (DCE-I and DCE-2) able to reduce VC at relative high rates is described in chapter 5. Cis-l,2-DCE is reduced at approximately 20-30 fold lower rates than VC. Our results suggest that these two enrichment cultures are able to gain energy from the reduction of lower chlorinated ethenes. When we performed these studies, no microorganisms had been isolated able to grow by the reduction of VC. However, recently He et al. (Nature. 2003, 424, 62-65) isolated Dehalococcoides strain BAVl, which is able to couple the reduction of DCE and VC to ethene to growth.

    Finally, the results obtained are combined with available literature data to obtain a state-of-the-art on chlorinated ethenes respiring microorganisms, the influence of alternative electron acceptors on these microorganisms and the role of H3 and H?-threshold values in halorespiration.

    Microbial diversity in archived agricultural soils; the past as a guide to the future
    Dolfing, J. ; Vos, A. ; Bloem, J. ; Kuikman, P.J. - \ 2004
    Wageningen : Alterra (Alterra-rapport 916) - 55
    bodembiologie - bodembacteriën - organische verbeteraars - mest - bodembeheer - organisch bodemmateriaal - geschiedenis - landbouwgronden - microbiële diversiteit - soil biology - soil bacteria - organic amendments - manures - soil management - soil organic matter - history - agricultural soils - microbial diversity
    Bacterial diversity and bacterially mediated processes are considered key to soil ecosystem functioning through decomposition and mineralization. However, there is a lack of understanding as to how activity and diversity of prokaryotic communities respond to changes in the environment. At present this issue is mostly addressed by real-time monitoring of long term field experiments, which is costly and slow. Using modern molecular methods we re-analyzed soil samples of up to 50 years old that have been stored in the Alterra soil archive TAGA. We showed that it is indeed possible to detect bacterial fingerprints in those samples and that fingerprints from different samples can be distinctly different, for example between fields that have or have not received organic amendments. These results are a promising first step towards unlocking the microbial information present in archived soil samples. This will help to assess the (likelihood of) changes in soil microbial diversity in response to environmental change (climate change) and human interference (fertilization) and to establish a reference condition and situation. This may further enable coupling to more functional assessments of soil functioning in standardized decomposition essays with the stored samples with known differences in diversity.
    Ondergronds veredelen, een braakliggend terrein : literatuur onderzoek naar de mogelijkheden voor veredeling op ontvankelijkheid voor biologische bestrijding, met name waardplant variatie voor effecten van rhizobacteria
    Balkema-Boomstra, A.G. - \ 2001
    Wageningen : Plant Research International - 20
    plantenveredeling - biologische bestrijding - rizosfeer - bodembacteriën - plant breeding - biological control - rhizosphere - soil bacteria
    Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions
    Ramirez-Saad, H.C. - \ 1999
    Agricultural University. Promotor(en): W.M. de Vos; A.D.L. Akkermans. - S.l. : Ramirez Saad - ISBN 9789058080660 - 119
    bodembacteriën - frankia - microbiële ecologie - benzoaten - soil bacteria - frankia - microbial ecology - benzoates

    Microbial Ecology studies aim to describe and assess the behavior and activity of microorganisms in their natural environments (Brock 1966). Nowadays it is clear that the large number of existing microorganisms has surpassed our capabilities to rapidly characterise them by traditional culturing methods. This has resulted in a poor understanding of the structure and composition of microbial communities. As an alternative, microbial communities can be described on the basis of 16S rRNA sequence diversity, without the bias-introducing step of cultivation.

    In the present thesis a molecular analysis is given of two ecosystems that harbour several uncultured bacteria. The first part of the thesis is focused on the detection and characterisation of Frankia in actinorhizal nodules and soil. Elucidation of the actual diversity within the family Frankiaceae was hampered by the inability to obtain isolates from all known actinorhizal plants. So far, the Nod +/Fix +Frankia symbionts in root nodules of plants from the families Coriariaceae, Datiscaceae, Rosaceae and Rhamnaceae (with exceptions reported by Carú 1993, Carú et al . 1990, and Carrasco et al . 1995) have resisted isolation. Best opportunities to characterise those uncultured endophytes require molecular methods that relay heavily on an easy and efficient technique to extract DNA from the respective actinorhizal nodules. Chapter 2 describes the techniques to isolate DNA from root nodules of different actinorhizal plants such as Casuarina sp, Alnus sp and Ceanothus sp. The procedure has also been successfully applied by Wolters et al . (1997b) in the minuscule ineffective nodules on Alnus glutinosa .

    Several attempts to characterise the uncultured endophytes from Coriaria sp. and Datisca sp. plants pointed on the one hand, to the presence in those actinorhizae of Frankia -related actinomycetes. This assumption was based mainly on the repeated isolation from those nodules of Nod -/Fix -Frankia -like strains (Hafeez 1983, Mirza et. al . 1994b, c). On the other hand, the effective (Fix +), non-isolated symbionts showed to be phylogenetically closely related (Mirza et al. 1994a), forming a separate lineage within the genus, in spite of the distant geographical distribution of the plants (Nick et al. 1992).

    The work described in Chapter 3 is focussed on the localisation and phylogenetic position of the nitrogen-fixing Frankia and Nod -/Fix -actinomycetes, both present in root nodules of the Mexican actinorhizal plant Ceanothus caeruleus . Application of the TGGE technique allowed localising the Nod -/Fix -actinomycete in the outer layers of the C. caeruleus nodules. Similar bacteria were also detected in Hippophaë rhamnoides nodules induced with soil inoculum that was collected in the vicinity of the former plant. The fact that a second nodule inhabitant was commonly present in these nodules containing recalcitrant endophytes may allow some speculations about their possible role in the symbiosis. However, it seems worthwhile to apply the same TGGE methodology to other actinorhizal nodules, even to those containing Frankia strains that are easy-to-isolate (i.e. Elaeagnus,Casuarina, Alnus spp.), since the detection of Frankia -related actinomycetes, in addition to the Fix +endophyte, would provide further evidence about the need for their presence. Coincidentally, the Nod -/Fix -isolates from Coriaria, Datisca and Ceanothus are phylogenetically related, pointing again to a certain specificity for their presence in the nodules. This relatedness has also been supported by analysis of low molecular weight RNA (i.e. 5S rRNA and tRNA's) using staircase electrophoresis (Velázquez et al. 1998).

    The 16S rDNA sequence from the non-isolated Fix +endophyte in C. caeruleus root nodules (Chapter 3), was the first full sequence obtained from a field-collected Ceanothus symbiont. Parsimony and phylogenetic distance analyses grouped it within the Dryas cluster that originally contained only the uncultured endophytes from Dryas , Coriaria and Datisca as proposed by Normand et al. (1996). Benson et al . (1996) redefined this cluster by adding other uncultured endophytes present in Ceanothus griseus (Rhamnaceae), Purshia tridentata and Dryas drummondii (Rosaceae) root nodules. Since the determined partial 16S rDNA sequences were almost identical, they suggested that the Frankia diversity from these actinorhizal plant families might be low. However, Clawson et al. (1998) demonstrated that Frankia isolates obtained from several genera within the Rhamnaceae (i.e. Talguenea , Colletia , Discaria, Retanilla and Trevoa ) were phylogenetically different than those in Ceanothus, grouping in the Elaeagnus cluster. These findings were consistent with morphological differences of the endophytes in planta , since the vesicles found in the Ceanothus symbionts resemble more to those in the Rosaceae, while all the latter host plants in the Rhamnaceae family have endophytes like those in Elaeagnus .

    The results reported in the first part of the thesis have demonstrated that TGGE and sequence analysis of 16S rDNA provide an accurate picture for the identification of recalcitrant endophytes in root nodules of actinorhizal plants. It has also been demonstrated that besides the N 2 -fixing endophyte, root nodules of C. caeruleus also harbour Frankia -related actinomycetes. Since these have also been observed in other actinorhizae, a further study is needed to understand the possible function of these co-symbionts.

    The work described in the second part of the thesis was addressing the changes occurring under chlorobenzoate stress in the soil bacterial community and other selected groups of bacteria present in peat soil collected from a natural Alnus glutinosa stand. A combination of culturing and non-culture based approaches was used for the assessment. Among the latter approaches, the possibilities offered by TGGE were exploited in several ways. Profiling of complex communities and subsequent analysis of specific bacterial groups has been one of the major applications of TGGE (Felske et al . 1996). With this approach, major population shifts induced by either 3CBA or 2,5DCB were detected in the uncultured bacterial community (Chapter 4). Although only the former compound was readily metabolised in soil, both xenobiotics promoted similar changes. Several bacterial populations were reduced or suppressed, while few others were enriched in time, as assessed by shifts in the TGGE banding patterns of the total bacterial community.

    To characterise the soil-enriched bacterial populations, 3CBA-degrading enrichment cultures were obtained and their composition was addressed by TGGE. Further isolation attempts were directed by this means to prove that the isolated strains were indeed the same enriched organisms as detected in soil. One of the enrichment cultures contained two of the soil-enriched bacteria as predominant components. Although isolation was not achieved, both bacteria were identified as belonging to the genus Burkholderia . The bacterial group detected as predominantly enriched in both spiked soils was not present in any of the enrichment cultures, suggesting that the microorganisms belonging to this groups are either unable to degrade 3CBA or not growing under the used culturing conditions. In any case their fitness to the soil conditions imposed by the addition of chlorobenzoates was high, but the mechanisms involved were not elucidated. These bacteria were also identified as Burkholderia by partial 16S rDNA sequence analysis (Chapter 4).

    The diversity (H) and the equitability (J) indices are important parameters used by ecologists to assess the species richness and the species evenness, respectively, within a community. As the estimation of such indices relies heavily on species definition and individuals enumeration, their application in microbial ecology studies is seldomly possible. Furthermore, assessment of H and J in uncultured bacterial communities must rely on the interpretation of community fingerprints, which should provide means to distinguish between species or operational taxonomic units (OTU), and to estimate their abundance. TGGE community profiling offers both possibilities, and the community changes occurring in the model soil system were evaluated with this original approach (Chapter 5).

    In addition, H and J indices were also estimated for the fluorescent pseudomonads group, a selected culturable fraction of the bacterial community. OTU recognition was addressed by using TGGE as a ribotype-fingerprinting technique for the isolated fluorescent pseudomonads. Estimation of H and J at the community level without culturing by TGGE profiling, and at the group level by a combination of culturing and TGGE ribotyping should allow to address and compare the population changes occurring, since the target molecule used in both TGGE was the same. Such comparison was only partially possible since most of the bands corresponding to the fluorescent pseudomonads could not be assessed in the community profiles. However, estimation of H and J indices indicated a clear reduction of species richness and individuals abundance in the uncultured community, which was related to the presence of chlorobenzoates in soil. Evaluation of population shifts by indexed values as H and J proved to be a useful means for analysing the community structure in time, and may be used to assess short and long-term responses of a bacterial community to environmental perturbations.

    Chapter 6 describes the changes in the total frankiae in soil and in the fraction of the population that is able to produce root nodules in Alnus glutinosa seedlings . Culture-independent approaches based on the most probable number concept were used, one in combination with a Frankia -specific PCR detection and another in combination with a plant-nodulation bioassay. After 15 days of incubation in the presence of chlorobenzoates both fractions of the soil Frankia populations were reduced in more than one order of magnitude, while the populations in the unspiked control soil were not affected. The results indicated that 3CBA and 2,5DCB both had a negative effect on the size of the native Frankia population from the used peat forest soil. This negative effect was also evident during in vitro experiments using Frankia strains isolated from Alnus sp. The presence of 1 mM 3CBA in the culture medium, in addition to the normal carbon source, resulted in reduction or suppression of biomass yield.

    The influence of Alnus glutinosa on the dechlorination of 3CBA by Pseudomonas sp. strain B13 was assessed in hydroponic cultures. It was expected that root exudates could enhance the dechlorination activity of Pseudomonas B13. When the bacteria were incubated in the presence of an alder plant, only a slight increase in the dechlorination rate of 3CBA was registered in comparison to the control without plant. The main observed effect in the alder plants appears to be a protection against 3CBA toxicity, as the alders inoculated with Pseudomonas B13 showed a better survival rate and grew more vigorously than the non-inoculated plants.

    The toolbox for microbial ecology studies is increasing constantly by means of developing new techniques or by adapting foreign tools into the field, such as indices to evaluate species diversity and eveness. Although the information provided by these community parameters facilitates comparisons and assessment of changes, their suitability to evaluate bacterial communities is still uncomplete. Estimation of diversity indices requires the recognition of bacterial species as discrete units, and this condition is far from real in natural environments. Species-independent approaches to evaluate diversity must be developed, that consider the bacterial diversity as a continuous range of phylogenetically related taxonomic units.

    In conclusion, the work described in the first part of the thesis strengthen the current phylogenetic division of the Frankiaceae, by adding new evidence supporting two of the already described clusters ( Dryas cluster, Nod -/Fix -cluster). Although the current taxonomic status of the latter cluster must be better evaluated in order to assess its pertinence to the genus Frankia . In addition, the common occurrence of Nod -/Fix -Frankia -like actinomycetes in nodules containing recalcitrant endophytes, mainly from the Dryas cluster, was also demonstrated for Ceanothus actinorhiza. In the second part, the suitability and versatility of molecular tools such as TGGE were demonstrated by their application in community profiling and estimation of bacterial diversity. Community changes occurring in stressed and unstressed soil systems were easily detected and assessed by this means.

    In addition, specific populations such as the Burkholderia -like bacteria that strongly reacted to the addition of chlorobenzoates to soil were further characterised. Moreover, TGGE was shown to be a fast ribotyping technique that may enable its use in combination with the community profiles to address shifts of specific groups within bacterial communities. It is tempting to suggest that this general approach will be of importance to direct the isolation of hitherto uncultured bacteria from soil.

    Response of predominant soil bacteria to grassland succession as monitored by ribosomal RNA analyses
    Felske, A. - \ 1999
    Agricultural University. Promotor(en): W.M. de Vos; A.D.L. Akkermans. - S.l. : S.n. - ISBN 9789058080110 - 113
    bodembacteriën - graslanden - nederland - ribosomaal rna - soil bacteria - grasslands - netherlands - ribosomal rna

    The research described in this thesis was aimed to provide insight into the effects of grassland succession on the composition of the soil bacteria community in the Drentse A agricultural research area. The Drentse A meadows represent grassland succession at different stages. Since 30 years particular plots have been taken out of intense agricultural production and were not fertilized anymore. However, the grass-vegetation was continuously removed once per year. This caused a progressive depletion of nutrients in the soil. In order to reveal the effect of grassland succession on soil microbes, the main bacteria in Drentse A grassland soils were identified by a molecular strategy based on detection and quantification of 16S rRNA. Instead of only using genomic 16S rDNA to reveal present sequences, we focused on rRNA to quantify the activity of the predominant bacteria. The ribosome is considered to be a useful marker for the overall metabolic activity of bacteria. In bacterial cultures the amount of ribosomes per cell has been found to be roughly proportional to growth activity. In our approach the activity is defined as total activity of one ribotype in relation to the bacterial community and not as activity per cell. Hence, bacteria of low activity per cell but extraordinary high cell number might be assessed as very active. Following direct ribosome isolation from soil, several different methods like RTPCR, separation of amplicons by temperature gradient gel electrophoresis (TGGE), different hybridization methods, cloning and sequencing were applied simultaneously to reveal the predominant 16S rRNA sequences for taxonomic identification. Quantitative dot blot hybridization with taxonspecific oligonucleotide probes revealed dominance of low G+C Gram-positives while other important groups appeared to be aProteobacteria and high G+C Gram-positives (Chapter 5). However, this approach did not demonstrate clear tendencies of community structure shifts by quantifying the rRNA of the major taxa. Therefore, a more sensitive method has been chosen, based on RT-PCR amplicons of bacterial 16S rRNA. The sequence-specific separation of these amplicons by TGGE reproducibly yielded characteristic band patterns from hundreds of soil samples (Chapter 3). Although the TGGE signals were very complex due to the high bacterial diversity in soil, different 16S rRNA fingerprints from a single plot were highly similar, while reproducible differences between plots of different history were observed. A parallel approach with PCRamplified genomic 16S rDNA led to similar results. The presence and activity of prominent bacteria in test fields of several hundreds m 2 were found to be quite homogeneous. Only one gram of soil was found to be representative for the predominant bacteria in large homogeneous grassland areas. After the high reproducibility of presence and activity was demonstrated for the main soil bacteria, representative TGGE fingerprints were compared to TGGE signals from a clone library of genes coding for 16S rRNA (Chapter 5). Cloned 16S rDNA amplicons matching the intense bands in the fingerprint were sequenced. The relationship of these sequences to those of cultured organisms of known phylogeny were determined. Approximately one half of the amplicons represented sequences closely related to those of cultured Bacillus -species, indicating that most of the active bacteria apparently belonged to the genus Bacillus . Other sequences similar to Gram-positive bacteria with high G+C-content were only rernotely related to those of cultured bacteria, as is illustrated by clone DA079 that could be affiliated to uncultured Actinobacteria from peat (Chapter 6). Another important group of sequenees was related to Proteobacteria, mainly the α-subclass. Several sequences could not be related to cultured organisms hut to the Holophaga/Acidobacterium - or the Verrucomicrobiales -cluster (Chapters 7 and 8).

    The parallel application of RT-PCR/TGGE and 16S rDNA-cloning to reveal the most abundant 16S rRNA sequences was found to be a powerful combination. The clone screening on TGGE was convenient and efficient, offering access to the almost complete 16S rRNA sequence. The subsequently performed V6-hybridization was a relatively simple approach to prove the identity of bands even in complex fingerprints (Chapter 6). The most predominant Bacillus -like ribotype DA001 in Drentse A grassland soils could also be detected by fluorescent whole-cell in situ hybridization (Chapter 10). Prominent rod-shaped cells of approximately 2 μm length could be identified in bacterial suspensions from soil with a multiple 16S rRNA probing approach. The specific DA001-signals represented about 5% of all microbial particles, which were visualized by the universal DNA-dye DAPI. Indeed, the sequences detected by the PCRbased methods represented abundant bacteria in soil. The most predominant Bacillus -like 16S rRNA sequence DA001 apparently originated from active, vegetative cells and not from endospores.

    The possibility to draw quantitative information about the microbial community from the complex TGGE fingerprints has been explored. A novel approach has been developed to quantify rRNA sequences in complex bacterial communities by multiple competitive RT-PCR and subsequent TGGE analysis (Chapter 4). The used primer pair (U968-GC and L1401) was carefully tested and found to amplify with the same efficiency 16S rRNAs from bacterial cultures of different taxa as well as the cloned 16S rDNA amplicons from soil samples. The sequence-specific efficiency of amplification was followed by monitoring the amplification kinetics via kinetic PCR. The primer-specific amplification efficiency was assessed by competitive PCR and RT-PCR, and identical input amounts of different 16S rRNAs were found to result in equal amplicon yields. We applied this method as multiple competitive RTPCR to TGGE fingerprints from soil bacteria to estimate the ratios of their 16S rRNAs (Chapter 9). This was done for different stages of grassland succession in the Dutch Drentse A area. The 16S rRNA amounts g -1 soil of 20 predominant ribotypes were monitored via multiple competitive RT-PCR in six plots of different succession stage. The 20 monitored 16S rRNA levels represented approximately half of all bacterial soil rRNA. The different Drentse A meadows, representing progressing stages of grassland succession, showed highly reproducible shifts of ribotype composition. In general, the rRNA levels were found to be doubled after the first years without fertilization. During the further progression of grassland succession the rRNA amounts were found to decline again. The 20 ribotypes showed remarkably different succession histories, causing the differences in TGGE fingerprints from different plots. While organic carbon and available nitrogen were declining during grassland succession, some bacteria were apparently suffering much more than the average. However, other bacteria showed an increased contribution to the bacterial rRNA pool, indicating that some bacteria could improve their position when less nutrients were available. The general increase in bacterial ribosomes in the first years after fertilization-stop was correlating to the increase of other parameters related to bacterial activity, i. e. carbon mineralization and microbial biomass. This suggested a true correlation between the total activity of bacterial communities in soil and the amount of ribosomes. This study provides extended information about uncultured bacteria in soil and describes the application and evaluation of several novel approaches in molecular microbial ecology. The following six conclusions are highlighting the major achievements and findings:

    1. Representative rRNA and rDNA fingerprints can be generated for homogeneous landscapes of large scale. This demonstrates that the often tiny sample size in molecular studies has not to be a limitation for microbial ecology. Nucleic acid extraction from small soil samples can be applied to characterize a several magnitudes larger environmental matrix. The composition of bacterial communities might be quite homogeneous for kilometers of grassland with heterogeneous vegetation and cultivation history. This conclusion is of general importance for molecular rnicrobial ecology and landscape ecology.

    2. The rRNA cycle (see Introduction, Fig. 2) for the predominant soil Bacillus recognized as ribotype DA001 has been completed. Its rRNA not only has been identified as predominant in the isolated fraction of soil ribosomes, but was also detected in an abundant type of bacterial eells by whole-cell hybridization to a fluorescently labeled, 16S rRNA-targeted oligonucleotide probe.

    3. The genus Bacillus appears to be dominant in the microbial community of Drentse A grassland soils. Uncultured members of the B. benzoevorans -line of descent are predominant among these bacilli. This group of Bacillus -ribotypes accounted for approximately 20% of all bacterial ribosomes in Drentse A soil. Such a predominant cluster of very closely related bacteria has never been observed before in soils. The reasons and circumstances of this special community composition remained unexplored.

    4. Prominent clusters of hitherto uncultured environmental bacteria were also detected in Drentse A soils. The Holophaga/Acidobacterium -cluster, the Verrucomicrobiales and a peat-related Actinobacteria-cluster had already been known from different locations all over the world. lt is the first time that these hitherto uncultured bacteria were identified as prominent contributors to the ribosome fraction in soil. Since these organisms apparently contain considerable amounts of intact ribosomes they are likely to be metabolically active. Some of these novel ribotypes belong to the most intense bands in the TGGE fingerprints, which may suggest a major role in environmental nutrient fluxes. This finding also contributes to the discussion about the unculturability of environmental bacteria, since based on the presented results it appears unlikely that the reason for their unculturability is a lack of viability. lt is more probable that suitable culture conditions are still not found yet. Since these bacteria are abundantly detected all over the world, future research should be aimed to attempt to culture them.

    5. A novel approach has been developed to quantify the predominant rRNA molecules of environmental bacteria communities. The multiple competitive RT-PCR allowed to quantify in a highly-specific way many different rRNA molecules within one assay. The RT-PCR-related possibilities of bias were investigated and excluded for the applied primer pair. Therefore, amplification by RT-PCR could be excluded as a major source of bias in this study. Other uncertainties are the selectivity of the applied primers and probes and the cell lysis efficiency. The selection of all the oligonucleotide probes and primers is based on only a few thousand 16S rRNA sequences of different length and quality. Although the available 16S rRNA sequence data are limited, the presenee of hitherto unknown bacteria with novel 16S rRNA sequences not matching the used primers is not indicated. The combined results of cloning, TGGE and dot blot hybridization, all achieved with different probes or primers, do not reveal possibly neglected groups of organisms. A serious bias caused by incomplete cell lysis may not be excluded. However, the majority of ribosomes originated from Gram-positives, indicating the lysis of bacteria with resistant cell walls. The possibility that highly resistant resting stages like endospores might have been missed is not relevant, since this study aimed to detect the most active bacteria.

    6. Multiple competitive RT-PCR revealed activity shifts for the predominant soil bacteria during Drentse A grassland succession. Some species responded to the nutrient depletion during grassland succession. Though the depleting nutritious matter and the changing vegetation, the overall impact of grassland succession did not cause a correspondingly drastic impact on the microbial community composition. Reproducible shifts of ribosome levels could be demonstrated, but the composition of the bacterial community remained remarkably stable. Evidence for major competition or replacement of species could not be found.

    Methane production and methane consumption: a review of processes underlying wetland methane fluxes.
    Segers, R. - \ 1998
    Biogeochemistry 41 (1998)1. - ISSN 0168-2563 - p. 23 - 51.
    lucht - hygiëne - luchtverontreiniging - methaan - broeikaseffect - opwarming van de aarde - bodembacteriën - moerasgronden - hoogveengronden - veengronden - wetlands - polders - air - hygiene - air pollution - methane - greenhouse effect - global warming - soil bacteria - swamp soils - bog soils - peat soils - wetlands - polders
    Potential rates of both methane production and methane consumption vary over three orders of magnitude and their distribution is skew. These rates are weakly correlated with ecosystem type, incubation temperature, in situ aeration, latitude, depth and distance to oxic/anoxic interface. Anaerobic carbon mineralisation is a major control of methane production. The large range in anaerobic CH_4:CO_2 production rates indicate that a large part of the anaerobically mineralised carbon is used for reduction of electron acceptors, and, hence, is not available for methanogenesis. Consequently, cycling of electron acceptors needs to be studied to understand methane production. Methane and oxygen half saturation constants for methane oxidation vary about one order of magnitude. Potential methane oxidation seems to be correlated with methanotrophic biomass. Therefore, variation in potential methane oxidation could be related to site characteristics with a model of methanotrophic biomass.
    Bepaling van afbraaksnelheden van organische stof in laagveen; ademhalingsmetingen aan ongestoorde veenmonsters in het laboratorium
    Vermeulen, J. ; Hendriks, R.F.A. - \ 1996
    Wageningen : DLO-Staring Centrum - 124
    organische verbindingen - bodemchemie - bodembacteriën - laagveengebieden - veengronden - kwantitatieve analyse - organic compounds - soil chemistry - soil bacteria - fens - peat soils - quantitative analysis
    De potentiële relatieve afbraaksnelheid (k-pot), de relatieve afbraaksnelheid onder anaërobe omstandigheden (k-an) en de invloed van de temperatuur op de afbraaksnelheid zijn voor drie veensoorten bepaald met een ademhalingsmeting. Verder is de stikstofmineralisatie bestudeerd. k-pot is afhankelijk van de veensoort en negatief gecorreleerd met de C-N-verhouding. k-an wordt sterk beonvloed door nitraat en sulfaat. De temperatuurinvloed (Q10) is groter naarmate de temperatuur lager is. Beneden 20 °C isde Q10 groter dan 2. Bij 0 °C is de afbraaksnelheid een kwart van die bij 10 °C. Ook in het stikstofarme veen treedt netto stikstofmineralisatie op.
    Cadmium and zinc interactions with a Gram-positive soil bacterium : from variable charging behavior of the cell wall to bioavailability of heavy metals in soils
    Plette, A.C.C. - \ 1996
    Agricultural University. Promotor(en): W.H. van Riemsdijk. - S.l. : Plette - ISBN 9789054855248 - 159
    bodembacteriën - grampositieve bacteriën - bodem - zink - cadmium - bodemgiftigheid - microbiële afbraak - biologische beschikbaarheid - soil bacteria - gram positive bacteria - soil - zinc - cadmium - soil toxicity - microbial degradation - bioavailability

    A detailed study is presented on the cadmium and zinc sorption to both isolated cell walls and intact, living cells of the Gram-positive soil bacterium Rhodococcus erythropolis A177. Acid/base titrations were performed on isolated cell wall material to characterize the type and amount of reactive sites on the cell wall. The proton binding was described with a three modal Langmuir-Freundlich equation, combined with a Donnan model to correct for the electrostatic interactions. Cadmium and zinc sorption to the isolated cell walls was reduced with increasing proton or calcium concentration. During the metal ion sorption, desorption of protons and calcium ions was observed. Calculations showed, that at high coverage with bivalent ions, charge reversal takes place. On the basis of the charging behavior, a competitive binding model, the NICA equation, was selected to describe the sorption data. The model is used to predict the sorption of cadmium and zinc to intact, living cells of the bacterium. The trends of sorption studies with intact cells at two exposure times suggest competitive interactions, not only for the adsorption of the metal ions to the cell wall, but also for the uptake into the cell. The impact of this competitive binding is reflected in the different levels of toxicity experienced for different pH conditions. A good correlation was found between cell wall adsorption and toxicity, indicating that for this organism, total body burden is a good indicator for potential effects, resulting from exposure to heavy metals. In combination with model descriptions of cadmium binding to a sandy and a clay soil, the influence of pH and calcium concentration on sorption to the bacterium when present in soil is predicted. Results show, that the impact of pH and calcium concentration is of the same order as the impact of soil type. It is quite obvious, that bioavailability is not only determined by the free metal ion concentration in solution, but that other parameters like the pH and the concentration of competing ions in solution play an important role as well.
    Survival of bacteria introduced into soil by means of transport by Lumbricus rubellus.
    Heijnen, C.E. ; Marinissen, J.C.Y. - \ 1995
    Biology and Fertility of Soils 20 (1995). - ISSN 0178-2762 - p. 63 - 69.
    aardwormen - bodembacteriën - bodemvruchtbaarheid - earthworms - soil bacteria - soil fertility
    Simulation of subsurface biotransformation
    Bosma, T.N.P. - \ 1994
    Agricultural University. Promotor(en): A.J.B. Zehnder; G. Schraa. - S.l. : Bosma - ISBN 9789054852216 - 136
    microbiële afbraak - geologie - biologie - bodembacteriën - organische verbindingen - organische scheikunde - microbial degradation - geology - biology - soil bacteria - organic compounds - organic chemistry

    Hydrophobic organic contaminants like DDT, Polychlorobiphenyls (PCB's) and polyaromatic hydrocarbons (PAH's), have been detected all over the world. They tend to accumulate in the atmosphere and in the soil as a result of their physical and chemical properties. Breakdown mainly proceeds by (photo)chemical reactions in the atmosphere and via microbial transformation in the soil. Microbial transformation can be viewed as part of the ecological process of decomposition, that is, the remineralization of organic material by biota. This Chapter discusses the ecological significance of biotransformation and the dependence of biotransformation rates on enviromnental conditions, and suggests ways to improve the effectiveness of biological soil remediation techniques.

    Contaminant cycling in ecosystems
    Chemicals are released into the environment by human activities. Normally, they enter the abiotic part of the ecosystem which may be viewed as a contaminant pool (Fig. S. 1). Biota take up contaminants directly from the abiotic environment e.g. via leaves or the skin, or ingest them by feeding on a lower trophic level. Organisms have systems at their disposal to excrete or detoxify contaminants. Excretion brings contaminants back to the contaminant pool, while detoxification results in a decontamination as indicated in Fig. S. 1.

    Plants and animals are not always able to detoxify or excrete contaminants after uptake. The inability of organisms to handle xenobiotic compounds may have several causes. One example is the absence of appropriate enzymes to transform the compounds, another the accumulation in (animal) fat tissue before excretion or enzymatic transformation has taken place. Contaminants accumulate in the food chain when organisms are not able to detoxify or excrete them. Accumulation is indicated by the use of different grey shades in Fig. S. 1.

    The population of "decomposers" (Fig. S. 1) is specialized in the uptake and conversion of all kinds of dead organic material, like for instance dead animals and plant debris. Decomposers are crucial for the functioning of ecosystems because they recycle nutrients to the nutrient pool. Contaminants which are accumulated in the tissue of organisms are recycled to the contaminant pool simultaneously. Some bacteria and fungi are able to detoxify and mineralize man-made organic compounds like chlorinated benzenes and polyaromatic hydrocarbons. Thus, they prevent their accumulation in the environment. These microorganisms may therefore be viewed as the "decontaminators" of ecosystems (Fig. S. 1), Many micro-organisms live in soil and ground water where hazardous compounds may accumulate. Microbial transformation is the only mechanism leading to the effective detoxification of such compounds. Therefore, it is of interest to know under which environmental conditions biotransformation is inhibited or stimulated. The potential of micro- organisms to transform contaminants under various environmental conditions is discussed in the following together with the factors governing exposure of micro-organisms to contaminants in soil and ground water.

    Potential of micro-organisms to transform organic contaminants
    The capacity of micro-organisms to detoxify anthropogenic chemicals under similar enviromnental conditions is variable among various habitats. This may be related to previous exposure of the micro-organisms to the compound under consideration. An adapted microflora capable of converting and mineralizing new compounds may evolve after a long exposure time. The microflora in a not pre- exposed environment may not be able to detoxify the same compound. Dichloropropene and 2,4-D (dichlorophenoxy acetic acid) are examples of pesticides that micro-organisms "learned" to transform. Degradation of these compounds in the field can be so rapid nowadays that their effectiveness as pesticide is strongly reduced. As a result, farmers have to apply considerably larger amounts of these pesticides than was necessary in the early times of their use.

    Many non-chlorinated organic compounds can be mineralized by aerobic bacteria. Well documented examples are simple aromatic compounds like benzene, toluene, and xylenes. ,More complicated aromatic structures like PAH's are also susceptible to aerobic degradation. Heavily chlorinated compounds are not readily degraded under aerobic conditions. However, anaerobic bacteria have a great potential to dehalogenate all kinds of such chemicals. Dehalogenation changes the environmental impact of the parent compounds considerably. Partly dechlorinated compounds are often more toxic and more mobile than the original compounds. The carcinogenic compound vinylchloride for example, may arise from the anaerobic dechlorination of tetra- and trichloroethene (PER and TRI). The anoxic transformation products are often biodegradable under aerobic conditions. The increased mobility of the more toxic products allows them to travel to aerobic environments where they
    can be mineralized. Thus, the anaerobic process of dehalogenation may be an important mechanism to initialize the complete mineralization of heavily chlorinated contaminants in the subsurface environment.

    Most of the information regarding the potential of micro-organisms to degrade organic contaminants is obtained from laboratory studies at 20°C. Studies carried out at temperatures down to 4°C, reveal only a slight temperature dependency of aerobic biotransformation rates. Anaerobic dehalogenation rates are reduced and intermediary dehalogenation products accumulate at lower temperatures. It seems that activities of aerobic micro-organisms involved in these processes are less dependent on temperature than those of anaerobes. Therefore, the aerobic removal rates of non- or partly halogenated compounds may be similar in summer and in winter in natural systems, while heavily halogenated compounds will tend to persist more in winter because of the reduced activity of anaerobes.

    A microscopic view of soil pollution and micro-organisms
    A picture of a versatile microbial community that is able to transform and mineralize a variety of hazardous organic compounds arises from the previous section. Nevertheless, biodegradable organic contaminants can persist in soil for decades. The microbial transformation rate of an organic compound is strongly affected by the potential uptake rate which is influenced by the transport rate to individual micro-organisms, The very slow in situ biotransformation is probably caused by the properties of the soil matrix surrounding the micro-organisms which reduces the transport rate. The microscopic spatial distribution of contaminants and micro-organisms will affect biotransformation rates in soil. This paragraph discusses how a spatial separation between micro- organisms and contaminants may develop in case of pollutions from point and non-point sources.

    Soil is polluted from point sources like for instance accidental spills and landfills (local pollution), or from non-point sources like atmospheric deposition and application of pesticides (diffuse pollution). The general characteristic of a local pollution is the presence of high contaminant concentrations in a small volume of soil (Fig. S.2A, upper part). Diffuse pollution is characterized by low contaminant concentrations over a wide area (Fig. S.2B, upper part). The lower part of Fig. S.2 schematically shows the local distribution of micro-organisms and contaminants in both situations. Bacteria are normally present inside soil aggregates. Low concentrations of contaminants flow around these aggregates in the case of diffuse pollution (Fig. S.2B). Local pollution initially contaminates pores around soil aggregates. The easily accessible part in wide pores may be biotransformed rapidly until nutrients become exhausted. This leads to a rapid growth of bacteria in the wide pores. Pollutants which are not biotransformed initially will diffuse into the aggregates. Thus, a situation arises with relatively high numbers of bacteria surrounding contaminated aggregates (Fig. S.2A). Degradation activity is drastically reduced as a result of spatial separation. A similar situation may arise when spots containing pure contaminant exist, where no biological activity is possible anymore. Hence, micro-organisms and contaminants are spatially separated both in the case of local and diffuse pollution. Biotransformation can only take place after diffusion of contaminant through the soil matrix to the micro-organisms.

    Computer calculations based on the concept presented in Fig. S.2 show that intra-aggregate processes of sorption and diffusion are of primary importance in determining the kinetics of biotransformation in soil. Effective diffusion rates in soil aggregates can be up to 1-10 orders of magnitude smaller than in water, depending on the characteristics of the soil matrix. As a consequence, biotransformation rates in different soils are subject to the same variation. When the diffusivity in soil aggregates is small, a steep concentration gradient is needed to maintain a flux of nutrients and contaminants that is sufficient to sustain microbial activity. As soon as the contaminant concentration drops below the value that is needed to maintain the gradient, biotransformation will stop. This threshold concentration is inversely proportional to the effective diffusion rate of contaminant. So, residual concentrations after biotransformation are expected to differ by many orders of magnitude, just like the biotransformation rates do.

    Optimization of bioremediation techniques to relieve limitation of biotransformation
    Limitation of biotransformation is not only the result of slow diffusion rates in soil, but may also be due to physiological or thermodynamic factors, to the presence of undissolved pollutants, or to the coupling of pollutant to soil organic matter via covalent bonds (bound residue formation). All these factors may result in reduced biotransformation rates. A strong association between the pollutants and the soil matrix especially develops at sites which have been polluted for years or decades already, Bound residue formation and extremely slow diffusion into small, highly tortuous pores have both been proposed as causes for this strong association. As a result, bioremediation is particularly difficult for these so-called "aged" pollutants (Fig. S.2A,

    Dissolution rates of undissolved pollutants may be enhanced by dispersing the pure component through the soil, and by the addition of surfactants that increase maximum dissolution rates. These methods have been shown to increase biotransformation rates in practice. However, surfactants do not dissolve bound residues which are covalently bound to organic matter. In addition, they do not increase diffusion rates in small, highly tortuous pores, The existence of bound residues and the extremely slow diffusion rates are causes for high residual concentrations that remain after bioremediation. Therefore, surfactants are not expected to decrease these residual concentrations.

    The possible application of procedures that enhance bound residue formation as means of bioremediation is disputed. It can be argued that pollutants which are present as bound residues are not hazardous anymore, having lost their specific chemical characteristics. Thus, they have also lost their biological activity. However, pollutants not only bind to the humus fraction of the soil, but also to dissolved or colloidal fractions of soil organic matter. This may lead to a mobilization of pollutants instead of the intended immobilization. In addition, dioxinlike products are formed when pollutants with phenolic or carboxylic groups bind to each other. The use of applications involving enhancement of bound residue formation requires that the possible hazards are better understood and that ways are provided to prevent them.

    Considerable residual concentrations will always remain after bioremediation of "aged" pollutions, due to strong sorption and incorporation in organic matter, unless special measures are taken to mobilize the pollutants. In an ex situ scheme, the soil may be pulverized to increase biotransformation rates and decrease residual concentrations. It is imaginable to remove the mobile fraction of pollutant in a relatively short time via a biological treatment during in situ remediation. The residual immobilized fraction which is trapped inside soil aggregates is biologically and chemically inactive. It should be sufficient to monitor and control pollutants that are slowly desorbing from the soil aggregates in an "after-care" phase. An approach may be to monitor the concentration level in the macropores continuously and to stimulate biotransformation by the addition of nutrients as soon as some critical level is reached. An alternative would be to apply a slow pump-and-treat method continuously, The after-care phase can be stopped as soon as total pollution concentrations in the soil are below acceptable limits.

    New pollutions have to be treated biologically as soon as possible to achieve optimal results because long contact times between pollutants and soil have a negative effect on the expected result of bioremediation. A possible strategy is to include biological soil treatment in human activities which almost inherently lead to soil pollution with organic chemicals. Thus, the establishment of a strong association between contaminants and soil can be prevented. This strategy has shown to be effective at tank stations where leaking of benzine or diesel is unavoidable.

    Introduction of specialized bacteria is used as a strategy to enhance the biotransformation of compounds that are not degraded by the indigenous microflora. The success of the addition of micro- organisms depends on their ability to reach the contamination, to survive, and to carry out the desired reaction. A better understanding and control of the transport of bacteria in soil and ground water will help to optimize techniques for bioremediation which employ introduced bacteria. Surface characteristics of bacteria and soil particles together with the ionic strength of the flowing water control the transport of bacteria under saturated flow conditions. The adhesion of bacteria to soil particles is positively correlated with the hydrophobicity of bacteria and the ionic strength of the flowing water. Hence, the ionic strength of the water in which bacteria are introduced can be used to control microbial transport and attachment. If a low ionic strength is used, bacteria may travel long distances and disperse around the point where they are introduced. On the other hand, a high ionic strength will generally stimulate the attachment of bacteria to the solid phase and may prevent bacteria from moving away from the polluted site.

    Concluding remarks
    Microbial transformation is required to achieve detoxification of hydrophobic organic contaminants that accumulate in soil. Micro-organisms can therefore be viewed as a subpopulation of the decomposers with a special function, namely detoxification of the environment. The effectiveness of microbial transformation can severely be reduced by the relative immobility of organic compounds in the soil matrix where micro-organisms live. Limitations resulting from slow diffusion can only be removed effectively during ex situ remediation, e.g. by pulverizing the contaminated soil. During a biological in situ treatment the bulk of contamination can be removed rapidly. The treatment should be followed by an aftercare period in which the possible leaking of the residual amount is monitored to be able to take measures if necessary.

    From an ecological stand-point, it can be argued that production and release rates of toxicants have to be smaller than in situ biotransformation rates to keep environmental pollution within acceptable limits. Treatment as close to the source as possible during the manufacturing and use of chemicals will be an important strategy to reach such a goal. The use of pesticides should be regulated such that the amount applied in a growth. season is completely transformed in situ in the same season.

    Discussions with Clay L. Montague were very helpful in the conception of the Synopsis.

    Contaminanten en micro-organismen: innig kontakt of Romeo en Julia?
    Schraa, G. ; Berg, R. van de - \ 1993
    Bodem 4 (1993). - ISSN 0925-1650 - p. 176 - 180.
    luchtverontreiniging - erosiebestrijding - microbiële afbraak - besmetters - biologische beschikbaarheid - bodembacteriën - bodemchemie - bodembescherming - bodemverontreiniging - air pollution - erosion control - microbial degradation - contaminants - bioavailability - soil bacteria - soil chemistry - soil conservation - soil pollution
    Microscopic counting and calculation of species abundances and statistics in real time with an MS-DOS personal computer, applied to bacteria in soil smears
    Bloem, J. ; Mullem, D.K. van; Bolhuis, P.R. - \ 1992
    Journal of Microbiological Methods 16 (1992). - ISSN 0167-7012 - p. 203 - 213.
    indicatorplanten - microscopen - microscopie - grondanalyse - bodembacteriën - bodembiologie - bodemfauna - bodemflora - computer software - programmeren - machine vision - programmeertalen - indicator plants - microscopes - microscopy - soil analysis - soil bacteria - soil biology - soil fauna - soil flora - computer software - programming - machine vision - programming languages
    Biological effects of plant residues with constrasting chemical compositions on plant and soil under humid tropical conditions
    Tian, G. - \ 1992
    Agricultural University. Promotor(en): L. Brussaard; B.T. Kang. - S.l. : Tian - ISBN 9789054850298 - 114
    mulchen - stoppelonderploegen - bodembacteriën - organische verbindingen - bodem - bodemchemie - bodemvruchtbaarheid - tropen - mulching - stubble mulching - soil bacteria - organic compounds - soil - soil chemistry - soil fertility - tropics

    A study on plant residues with contrasting chemical compositions was conducted under laboratory, growth chamber and humid tropical field conditions to understand the function of the soil fauna in the breakdown of plant residues, the cycling of nutrients, in particular nitrogen, and the performance of maize as a test crop. Leaves from ten agroforestry and fallow plant species with a wide range of chemical compositions (different C/N ratios and contents of lignin and polyphenols) were selected for laboratory incubations. Of the ten species, prunings from Acioa barteri , Gliricidia sepium and Leucaena leucocephala , maize ( Zea mays ) stover and rice ( Oryza sativa ) straw were selected for field and growth chamber experiments.

    Laboratory incubation showed that N mineralization was significantly correlated with initial N, polyphenol and lignin content of plant residues. Litterbag studies in the field showed that decomposition was also correlated with the activity of soil fauna. In the field the effect of polyphenols on the degradation of plant residues was less prominent than in the laboratory. In both growth chamber and field trials the breakdown of plant residues and nutrient release were enhanced in the presence of earthworms and millipedes.

    Application of plant residues increased populations of earthworms and ants. Stepwise regression illustrated populations of earthworms were negatively correlated with lignin:N ratio; populations of ants were positively correlated with N content of plant residues. Application of high C/N ratio and lignin content plant residues attracted termites. Millipedes were not influenced by the quality of plant residues.

    Addition of plant residues as mulch ameliorated the soil microclimate by lowering soil temperature and maintaining soil moisture. A strong mulching effect on the soil microclimate was associated with application of plant residues with high C/N ratio and lignin content.

    Increase in the amount of mineral N in the soil solution was recorded with application of plant residues with "high" or "low quality", but probably caused by different mechanisms. The N contribution of "high quality" materials mainly originated from their decomposition, whereas mulching effects on microclimate, which presumably promoted the degradation of soil organic matter, mainly accounted for the N contribution of "low quality" materials. There is a need to both consider the direct and indirect contributions of plant residues applied as mulch.

    Markedly improved crop performance was achieved with the application of plant residues with "high" or "low quality". Materials with "intermediate quality" had no significant impacts.

    Results of the study indicate the possibility to influence nutrient cycling by manipulation of soil faunal activity.

    It is concluded that a keen choice of plant residues in terms of nutritional effects and mulching-induced effects on soil microclimate is a prerequisite for the application of soil fauna-mediated decomposition towards synchronization of soil nutrient supply and crop nutrient demand.

    Population dynamics of bacteria introduced into bentonite amended soil
    Heijnen, C. - \ 1992
    Agricultural University. Promotor(en): N. van Breemen; J.A. van Veen. - S.l. : Heijnen - 119
    bodembacteriën - stikstofbindende bacteriën - symbiose - rhizobium - bodemstructuur - verbetering - zand - bodemverbeteraars - natuurlijke hulpbronnen - kleimineralen - bentoniet - pseudomonas - soil bacteria - nitrogen fixing bacteria - symbiosis - rhizobium - soil structure - improvement - sand - soil conditioners - natural resources - clay minerals - bentonite - pseudomonas

    Bacteria have frequently been introduced into the soil environment, e.g. for increasing crop production or for biological control purposes. Many applications require high numbers of surviving organisms in order to be effective. However, survival of bacteria after introduction into soil is generally poor, and numbers of introduced bacteria have been known to decrease from 10 9to approximately 10 3cells/g soil in 25 days. Thus, if bacteria are to be used as effective microbial inoculants to, a means to increase survival levels in soil needs to be found.

    Survival of Rhizobium leguminosarum biovar trifolii introduced into loamy sand was found to be greatly enhanced by amendments of bentonite, in amounts of 5 or 10%, to the soil. Bentonite appeared to offer introduced bacteria protection against protozoan predation, resulting in increased bacterial survival levels in bentonite amended soil as compared to unamended soil.

    Also in liquid cultures, protozoan activity was strongly hampered by the presence of bentonite, thereby also improving the survival of Rhizobium. Bentonite did not release any substances toxic to protozoa in liquid cultures, and presumably this would not occur in the soil environment either. Bentonite toxicity could therefore not explain the increased survival levels of bacteria introduced into bentonite amended soil. It was suggested that, in liquid cultures, bentonite clay increased the minimum level of bacteria for effective predation by protozoa.

    Changes in soil structure as a result of bentonite additions could explain the observed increases in bacterial survival levels. A mathematical relationship was found describing the log numbers of introduced rhizobia surviving in soil samples after an incubation period of 57 days using 3 pore size classes. Pores with necks < 3 μm and between 3 and 6 μm positively affected survival levels. These pores apparently were large enough to allow bacteria to enter, but were too small to be accessible to predating protozoa. Pores with necks between 6 and 15 μm had a negative influence on rhizobial survival levels, because bacteria situated inside these relatively large pores could be reached and predated upon by protozoa. Therefore, pores < 6 μm were found so serve as protective microhabitats for bacteria introduced into soil. A larger number of such protective microhabitats in bentonite amended loamy sand than in unamended loamy sand could explain the observed increase in survival levels of bacteria introduced into bentonite amended soil. The colonization potential of protective microhabitats was suggested to be determined largely by pore shape and the continuity of the water-filled pore system. Increased numbers of protective microhabitats (pores < 6 μm) in bentonite-amended soil as compared to unamended soil were demonstrated visually by micro-morphoiogical studies using Cryo Scanning Electron Microscopy.

    The effectiveness of bentonite was strongly determined by the way in which the clay and the inoculum were added to the soil. When a bentonite suspension and bacteria were mixed together prior to inoculation, the clay offered more protection against predation than when bentonite powder and bacteria were added separately. This suggested that when the protective agent was present at the site of introduction, a more efficient use of the clay could be made, resulting in enhanced survival levels.

    Apart from survival, bentonite additions to soil also influenced bacterial respiration. The cumulative amount of CO 2 respired by rhizobia introduced into sterile bentonite- amended loamy sand was significantly higher than in unamended loamy sand. Carbon was used more efficiently during growth in bentonite-amended than in unamended loamy sand. The maintenance respiration of rhizobial cells was not influenced by the presence of bentonite clay. The growth rate of rhizobia introduced into sterile soil was increased by the presence of bentonite.

    Pseudomonas fluorescens was also found to survive at higher levels in bentoniteamended than in unamended soil, suggesting the bentonite effects were not limited to Rhizobium only. Pseudomonas fluorescens was used to study root colonization by bacteria introduced into bentonite amended soil. A rhizosphere effect (i.e. the occurrence of higher cell concentrations in rhizosphere soil than in bulk soil) was observed both in the absence and in the presence of bentonite clay, but it was less pronounced in the latter case. This finding suggested that bacteria were physically hindered by bentonite, making it more difficult to invade the immediate root environment. However, protection against predation by bentonite enhanced survival to such an extent, that overall survival in the rhizosphere was still higher in bentonite amended loamy sand than in the unamended soil.

    It can be concluded from this thesis that soil structure, and especially the pore size distribution of the soil is a key factor determining the survival, chances of bacteria introduced into soil. Application of introduced bacteria for e.g. biological control will probably stand a larger chance of being successful in soils with relatively high numbers of pores < 6 μm However, it is unlikely that bentonite will ever be applied to soil in amounts of e.g. 5%, because of the large impact of bentonite additions on, for example, the moisture characteristics of the soil. However, the knowledge obtained on the importance of the pore size distribution of a soil, and the fact that the she of introduction of the bacteria will largely determine survival chances, will be of great importance for the future development of successful carrier materials for introducing bacteria into soil.

    Molecular mechanisms of adaptation of soil bacteria to chlorinated benzenes
    Meer, J.R. van der - \ 1992
    Agricultural University. Promotor(en): A.J.B. Zehnder; W.M. de Vos. - S.l. : Van der Meer - 100
    bodembacteriën - pseudomonas - derivaten - benzeen - chloride - hexachloorbenzeen - micro-organismen - genetica - heritability - adaptatie - soil bacteria - pseudomonas - derivatives - benzene - chloride - hexachlorobenzene - microorganisms - genetics - heritability - adaptation - cum laude

    The pollution of our environment with a large number of different organic compounds poses a serious threat to existing life, since many of these chemicals are toxic or are released in such quantities that exceed the potential of biological detoxification and degradation systems. Bacteria and other microorganisms play an essential role in the breakdown of xenobiotic compounds. Microbes use these compounds as carbon and energy source and metabolize them to harmless endproducts. However, not all compounds are easily metabolized and some structures resist the action of existing enzyme systems in bacteria. Nevertheless, bacterial species have been isolated which have overcome these metabolic barriers and completely metabolize chemicals that were previously considered to be persistent.

    The project of this thesis was initiated to study the genetic mechanisms in bacteria that cause adaptation to use xenobiotic compounds as novel growth substrates (see Chapter I for a review). The work presented here mainly focused on one class of compounds, i. e. lower chlorinated benzenes such as dichlorobenzenes (DCB) and 1,2,4- trichlorobenzene (1,2,4-TCB). These compounds were known to be very resistant to biodegradation by bacteria. A number of bacterial species was isolated by enrichment techniques which were able to use DCB's and/or 1,2,4-TCB as sole carbon and energy source for growth. One of these bacteria, Pseudomonas sp. strain P51, was investigated further in this study. We have obtained strong evidence that the pathway for chlorobenzene metabolism arose as a consequence of the unique combination of two gene clusters, each specifying part of the complete pathway. These individual gene clusters are not uncommon and probably exist separately in different bacteria. Our results suggest that one of the gene clusters is contained in a novel transposable element that may have been acquired by strain P51 and integrated into a catabolic plasmid that already contained the other gene cluster. A further fine-tuning of the new pathway may have occurred through specialization of individual enzymes towards novel metabolic intermediates and by changes in the regulatory system in response to novel inducer molecules.

    The degradation of DCB's and 1,2,4-TCB was studied at concentrations between 10 μg/l and 1 mg/l in soil percolation columns filled with sediment of the Rhine river, which in some cases were inoculated with Pseudomonas sp. strain P51 (Chapter 2). In the inoculated columns, DCB's and 1,2,4-TCB were instantly degraded. Strain P51 remained viable in the column as long as the chlorinated benzenes were fed in the influent. Interestingly, minimal concentrations of the chlorinated benzenes were measured in the effluent of the columns, independently of the influent concentrations used (6 ± 4 μg/l for 1,2-DCB; 20 ± 5 μg/l for 1,2,4-TCB; more than 20 μg/l for 1,3-DCB and 1,4-DCB), which could not be lowered by additional inoculations with strain P51. The native microbial population in the noninoculated columns adapted to degrade 1,2-DCB after a lag phase of about 60 days, and was then able to remove a concentration of 25 μg/l of 1,2-DCB in the influent to less than 0.1 μg/l.

    Detailed genetic studies were carried out with Pseudomonas sp. strain P51 to characterize the genetic determinants for chlorobenzene metabolism. A large plasmid of 110 kilobase-pairs (kb) (pP51) could be isolated from cells that were cultivated on 1,2,4- TCB (Chapter 3). This plasmid could be cured from the strain by successive inoculations on non-selective media, rendering the strain incapable of metabolizing chlorinated benzenes. Subsequent cloning and deletion experiments in Escherichia coli, Pseudomonas putida, and Alcaligenes eutrophus showed that two regions on plasmid pP51 were responsible for chlorobenzene metabolism. Expression studies in E. coli revealed that a 5-kb region encoded the activity to convert 1,2,4-TCB and 1,2-DCB to 3,4,6-trichlorocatechol and 3,4-dichlorocatechol, respectively. This activity was determined using whole cell incubations, and in analogy with other described catabolic pathways it was proposed that the activity was caused by a chlorobenzene dioxygenase multienzyme complex and a dehydrogenase (encoded by tcbA and tcbB, respectively). Separated from the chlorobenzene dioxygenase gene cluster by approximately 6 kb a region was located which contained the genes for the conversion of chlorocatechols. Different DNA fragments of this region of pP51 were cloned in expression vectors in E. coli, P. putida and A. eutrophus. Both P.putida KT2442 and A. eutrophus JMP222 could be complemented for growth on 3-chlorobenzoate by a 13-kb fragment of pP51, which indicated that a functional pathway for degradation of chlorocatechols was encoded on this fragment. Enzyme activity measurements and transformation reactions with 3,4-dichlorocatechol in cell extracts of E. coli harboring cloned pP51 DNA fragments showed the activity of three enzymes, chlorocatechol 1,2-dioxygenase (catechol 1,2-dioxygenase II), chloromuconate cycloisomerase (cycloisomerase II), and dienelactone hydrolase II. The genes encoding these activities were designated tcbC, tcbD, and tcbE, respectively, and their deduced gene order was found to be tcbC-tcbD- tcbE. It was thus proposed that 3,4-dichlorocatechol was converted via a chlorocatechol oxidative pathway (or modified ortho cleavage pathway), similar to that described in Pseudomonas sp. strain B 13 and A. eutrophus JMP134 , leading finally to the formation of 5-chloromaleylacetate. The release of one chlorine atom from 3,4- dichlorocatechol was shown to take place spontaneously during lactonization in the cycloisomerization reaction.

    The genes of the chlorocatechol oxidative pathway of strain P51 are organized in a single operon, comprising a region of 5.5 kb, which was fully sequenced and contained five large open reading frames (Chapter 4). The gene products of the different open reading frames were analyzed by subcloning appropriate pP51 DNA fragments in E. coli expression vectors. Expression studies confirmed the previously determined gene order and could attribute three open reading frames to the gene loci tcbC, tcbD, and tcbE, respectively. In between tcbD and tcbE an 1,022 bp open reading frame was present (ORF3), but we could not detect any protein encoded by this ORF. Immediately downstream of tcbE another ORF was found, designated tcbF, which encoded a 38 kDa protein. Until now, no clear function has been attributed for the tcbF gene product. The tcbCDEF genes and their encoded gene products showed high (50.6% - 75.7%) homology to two other chlorocatechol oxidative gene clusters, clcABD of P.putida (pAC27) and tfdCDEF of A. eutrophus JMP134(pJP4). Furthermore, a homology of 22% and 43.9% was found of TcbC and TcbD to CatA and CatB, respectively, the catechol 1,2-dioxygenase and cycloisomerase of the β-ketoadipate pathway of Acinetobacter calcoaceticus. This suggests that the chlorocatechol oxidative pathway originated from other, more common, metabolic pathways. Despite the strong DNA and amino acid sequence homology of the genes and enzymes of the chlorocatechol oxidative pathways, the substrate range of the pathway enzymes from the three organisms differed subtly. This was demonstrated for the chlorocatechol 1,2- dioxygenases TcbC, ClcA, and TfdC. In contrast to ClcA and TfdC, which showed a high relative activity for 3,5-dichlorocatechol, TcbC exhibited a strong preference for 3,4- dichlorocatechol and a weak affinity for the 3,5-isomer. This suggested that the tcb -encoded pathway enzymes had become specialized for intermediates (i.e. 3,4- dichlorocatechol) which arise in the metabolism of the novel compound 1,2- dichlorobenzene. Different genetic mechanisms may cause the divergence of genes and allow a specialization of encoded proteins (see also Chapter 1). Recently it has been proposed that slippage of short sequence repetitive motifs and subsequent mismatch repair would be the major driving force for rapid evolutionary divergence, rather than single base-pair substitutions. Detailed DNA sequence comparisons between the chlorocatechol 1,2-dioxygenase genes tcbC , clcA , and tfdC gives evidence for slippage of short sequence repetitions in regions of strong divergence in amino acid sequence.

    The transcription of the tcbCDEF operon was found to be regulated by the gene product of tcbR, a gene located upstream of and divergently transcribed from the tcbC gene. The tcbR gene was characterized by DNA sequencing and expression studies in E. coli and appeared to encode a 32 kDa protein (Chapter 5). The activity of the tcbR gene was analyzed in P.putida KT2442 harboring the cloned tcbR and tcbCDEF genes by determining the activity of the chlorocatechol 1,2-dioxygenase TcbC during growth on 3-chlorobenzoate. Strains of P.putida KT2442, which carried a frame shift mutation in the tcbR gene, could no longer induce tcbC expression during growth on 3-chlorobenzoate, suggesting that TcbR functions as a positive regulator of tcbC expression. A region of 150-bp is separating tcbR and tcbC, the first gene of the tcbCDEF cluster, and contains the expression signals needed for the transcriptional activation of tcbCDEF by the tcbR gene product. The transcriptional start sites of tcbR and tcbC were determined by primer extension analysis and this showed that the two divergent promoter sequences of the genes overlap. Protein extracts of both E. coli overproducing TcbR and of Pseudomonas sp. strain P51 showed specific DNA binding to this 150-bp region. TcbR probably regulates tcbCDEF expression and autoregulates its own expression, by binding the DNA region containing the promoters of tcbC and tcbR. It is likely that an inducer molecule interacts with TcbR, which may cause alterations or partially unwinding of the bound region and stimulation of RNA polymerase to start transcription of the tcbCDEF operon. Amino acid sequence comparisons indicated that TcbR is a member of the LysR family of transcriptional activator proteins and shares a high degree of homology with other activator proteins involved in regulating the catabolism of aromatic compounds, such as CatM, CatR and NahR. Detailed studies have recently been carried out to determine the precise interaction of TcbR with its operator region by DNasel footprinting. It would be interesting to see if in analogy with the specialization of TcbC, TcbR has diverged from a more common regulator protein such as CatM or CatR, and became specialized in recognizing chorinated inducer molecules.

    DNA sequence analysis of the start of the chlorobenzene dioxygenase cluster revealed the presence of an insertion element, IS 1066 (Chapter 6). An almost exact copy of this element, IS 1067, was discovered on the other side of this gene cluster, although oriented in an inverted position. Thus, the complete genetic element formed by IS 1066, the tcbAB gene cluster, and IS 1067, resembled a composite bacterial transposon. The functionality of this transposon, which was designated Tn 5280 , was established by inserting a 12-kb Hin dIII fragment of pP51 containing Tn 5280 , marked with a kanamycin resistance gene in between the IS-elements, into the suicide donor plasmid pSUP202 followed by conjugal transfer to P.putida KT2442. Analysis by DNA hybridization of transconjugants with acquired kanamycin resistance showed that Tn 5280 had transposed into the genome of this strain at random and in single copy. The insertion elements IS 1066 and IS 1067 were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and Agrobacterium rhizogenes, and were distantly related to IS 630 of Shigella sonnei. The presence of the tcbAB genes on Tri 5280 suggested a mechanism by which a chlorobenzene dioxygenase gene cluster was mobilized as a gene module by the mediation of IS-elements. This gene module was then joined with the chlorocatechol gene cluster to form the novel chlorobenzene pathway.

    To obtain more information on the distribution of chlorobenzene degradation genes in the environment, different methods were applied which were based on DNA- DNA hybridization with gene probes derived from chloroaromatic metabolism (Chapter 7). A number of bacterial strains which were isolated by selective enrichment from soil samples for growth on chloroaromatic compounds .was screened for the presence of catabolic plasmids. Hybridization of these plasmid-DNA's with DNA fragments of the tcbAB or tcbCDEF genes revealed a class of plasmids which were identical or homologous to plasmid pP51 of strain P51. In other experiments soil microorganisms were directly extracted from soil samples, plated on nonselective media and screened by DNA-DNA colony hybridization for the presence of catabolic genes with a set of probes for three chlorocatechol 1,2-dioxygenase genes (tcbC, clcA, and tfdC). Positively reacting colonies were obtained under selective conditions with a frequency of 1 to 5 per 2000, which indicated that in the soil samples microorganisms were present which contained DNA sequences homologous to the used probes. However, from additional screening and hybridization experiments of these positively reacting colonies it became clear that some of these were false positives. Furthermore, positive strains were lost easily during transfer from the original agar plates due to the heterogeneity in colony types of the different soil microorganisms. In a third method the variation of chlorocatechol 1,2-dioxygenase genes among soil microorganisms was analyzed by amplifying total DNA from soil samples in the polymerase chain reaction, which was primed with degenerate oligonucleotides derived for conserved regions in tcbC, clcA, and tfdC. Discrete amplified fragments were obtained in this manner, which were cloned and analyzed by hybridization and DNA sequencing. We found six different types of fragments which had the expected size, only one of which was related significantly to the chlorocatechol 1,2-dioxygenase (and in fact was identical to the tcbC- type). This indicated that it was possible to detect and isolate chlorocatechol 1,2-dioxygenase sequences from soil DNA although the selectivity of the amplification reaction was relatively low.

    In this study, we have entered a field of microbial research which will have continuing evolutionary and environmental interest. A detailed genetic characterization of bacteria which adapted to use xenobiotic compounds as novel growth and energy subsrates, suggested different mechanisms by which novel metabolic pathways evolve in bacteria. Our results presented evidence for i) a specialization of enzyme systems and ii) an exchange and combination of pre-existing gene modules. Still we do not know what the capacity of microorganisms present in the natural environment is to adapt rapidly to metabolize xenobiotic substrates, nor do we know how and which environmental factors influence genetic adaptation. Astonished by the diversity of genetic mechanisms displayed in bacteria which govern evolutionary change, we shouldn't be surprised to find mechanisms which direct and regulate genetic adaptation in response to changing environmental conditions.

    Influence of different application rates of nitrogen to soil on rhizosphere bacteria
    Liljeroth, E. ; Schelling, G.C. ; Veen, J.A. van - \ 1990
    Netherlands Journal of Agricultural Science 38 (1990)3A. - ISSN 0028-2928 - p. 255 - 264.
    toedieningshoeveelheden - kunstmeststoffen - hexaploïdie - stikstof - populatiedynamica - bodembacteriën - triticum aestivum - tarwe - application rates - fertilizers - hexaploidy - nitrogen - population dynamics - soil bacteria - triticum aestivum - wheat
    Onderzoek naar de bacterieele populaties in de rhizosfeer van tarwe bij verschillende stikstofregimes. De stikstofgift (250 mg N en 50 mg ␛ per plant) werd gegeven in een eenmalige of gesplitste gift, waarbij steeds kleinere hoeveelheden werden gegeven. De verschillende giften maken het mogelijk om de bacterieele populaties te bestuderen in relatie tot de stikstofconcentraties in de wortels en in de bodem bij overeenkomstige biomassaproduktie
    Structuurveranderingen door toevoeging van kleimineralen aan lemig zand
    Schoonderbeek, D. ; Schoute, J.F.T. - \ 1990
    Wageningen : Staring Centrum (Rapport / Staring Centrum 120) - 23
    keileemgronden - kleimineralen - verbetering - leemgronden - pedologie - zand - bodembacteriën - bodemverbeteraars - bodemmicromorfologie - bodemstructuur - boulder clay soils - clay minerals - improvement - loam soils - pedology - sand - soil bacteria - soil conditioners - soil micromorphology - soil structure
    Distribution and population dynamics of Rhizobium sp. introduced into soil
    Postma, J. - \ 1989
    Agricultural University. Promotor(en): A.J.B. Zehnder; J.A. van Veen. - Wageningen : Wageningen UR Library - 121
    rhizobium - bodeminoculatie - stikstofbindende bacteriën - knobbeltjes - symbiose - microbiologie - landbouw - populatiedynamica - bodembacteriën - rhizobium - soil inoculation - nitrogen fixing bacteria - nodules - symbiosis - microbiology - agriculture - population dynamics - soil bacteria

    In this thesis the population dynamics of bacteria introduced into soil was studied. In the introduction, the existence of microhabitats favourable for the survival of indigenous bacteria is discussed. Knowledge about the distribution of introduced bacteria over such microhabitats, however, is scarse. Nevertheless, it was hypothesized that upon introduction, bacteria reach other microsites in soil than bacteria which are already present for some time, thereby influencing the survival of introduced organisms. Methods to study the distribution of introduced bacteria in soil, as well as the effect of their distribution on the population dynamics, were assessed. A model organism, Rhizobium leguminosarum biovar trifolii and two different soils, a loamy sand and a silt loam, were used for this purpose.

    Two methods for the enumeration of bacteria introduced into soil were compared (Chapter 2). Although immunofluorescence was a very promissing method at the moment we started our work, selective plating proved to be more suitable for the enumeration of low numbers of introduced bacteria, since it had a lower detection limit than immunufluorescence. In addition, selective plating did not depend on flocculation processes which were shown to influence significantly the results obtained with the immunofluorescence technique. Moreover, only cells able to divide were counted. However, with the immunofluorescence technique we were able to determine cell lengths and we detected that the length of cells which were grown in a rich medium decreased after their introduction into soil.

    To study the micro-distribution of bacteria in soil, different fluorochromes were tested on their ability to stain bacteria in thin sections of undisturbed soil samples. Calcofluor white M2R in combination with acridine orange was successfully applied for the detection of bacteria in thin soil sections (Chapter 3). However, specific staining of the introduced rhizobia with conjugated antiserum was not successful. Therefore, an alternative method for the assessment of the distribution of introduced bacteria, a soil washing procedure, was used in Chapters 4, 5 and 6. With this method, free occuring bacteria and bacteria associated with soil particles or aggregates>50 μm were distinguished.

    The bacterial distribution through soil could be manipulated by inoculating soils at different initial moisture contents. At a lower initial moisture content, only the narrowest pores are filled with water, so that inoculated rhizobial cells will reach narrower pores when they are transported passively by the waterflow. At a higher initial moisture content, water already present in the narrower pores prevent the introduced cells from entering these pores. With such an inoculation procedure, rhizobial cells were found to be associated to a larger extent with soil particles when soils were inoculated at lower initial moisture contents. In natural soils, this resulted in an improved survival of rhizobia during at least 100 days after inoculation (Chapter 4). Moreover, the number of particleassociated cells decreased less than the number of free occuring cells in natural soil. It was concluded that rhizobial cells associated with soil particles or aggregates>50 μm occupied a more favourable microhabitat than free occuring cells. In sterilized soil, numbers of both particleassociated and free occuring cells increased and the initial differences in distribution did not result in different final population levels (Chapter 5). Therefore, it was concluded that the microhabitats in natural soil rendered protection to biotic rather than to abiotic factors.

    The influence of competitors and predators on the distribution and population dynamics of rhizobium was studied by the addition of specific groups of organisms to sterilized soils (Chapter 5). Previous to inoculation with rhizobia, sterilized soils were recolonized with several bacterial isolates which were obtained from these soils and part of the soil portions were inoculated with a flagellate precultered on rhizobial cells. In the presence of flagellates, which predate on bacteria, a higher percentage of particle-associated rhizobial cells was present than in the absence of flagellates. In recolonized soils, i.e. in the presence of competitors, the percentages of particle -associated rhizobial cells were lower than in soils that were not recolonized previous to inoculation. Thus, the presence of competitors made it more difficult for rhizobial cells to colonize the microsites where they can be associated with soil particles or aggregates. The total number of rhizobial cells was influenced only little (silt loam) or not at all (loamy sand) by the competitors or by the addition of flagellates alone. However, when both competitors and predators were present, numbers of rhizobial cells decreased drastically. This synergetic effect was explained by hypothesizing that after the predation of accessible bacterial cells by the flagellates, the regrowth of rhizobial cells will be limited by the presence of competitive microorganisms in many of the favourable microhabitats.

    The association of rhizobial cells with soil particles may be the result of enclosure in pores or attachment to soil surfaces of rhizobia. The role of attachment was studied with a R. leguminosarum strain and three Tn5 mutants which were altered in their cell surface properties (Chapter 6). Although the importance of association with soil particles or aggregates was affirmed, the results gave no evidence that attachment to soil particle surfaces was an important factor for the survival of introduced cells.

    The final population level of introduced rhizobia was studied in more detail by inoculating sterilized and natural soils with different inoculum levels (Chapter 7). In sterilized soils, populations reached, independent of the inoculum density, a final level which was suggested to represent the carrying capacity of the soils in terms of available habitable pore space, moisture and substrate for survival of the bacteria. In natural soil, however, the survival levels were dependent on the inoculum density. In this case, the chances of introduced cells to reach favourable microhabitats, determined the survival level of the entire population.

    In all experiments final population levels in natural and in sterilized soils were higher in the silt loam than in the loamy sand (Chapter 2-8). Pore space which is suitable for bacteria to survive (=habitable pore space) or which protects bacteria from predation (=protective pore space) was estimated for both soils. The occupancy by bacteria was in all cases lower than 0.5%, so that no serious space limitation could be expected. Therefore, the larger water-filled pore volume at the water potential used (pF 2) in the silt loam as compared to the loamy sand, could not explain the differences in population sizes. In sterilized soil substrate availability was suggested to determine the final population level. In natural soil, however, the survival of rhizobial cells was suggested to be dependent on the number of introduced bacteria that reached the protective pore space (Chapter 4 and 7).

    In this thesis it is shown that the soil washing procedure is useful for the study of the distribution of introduced bacteria. Immediately after introduction, only few bacteria were associated with soil particles. The number of particle-associated bacteria decreased less pronounced than the number of free occuring bacteria, giving evidence that the distribution of introduced bacteria in soil is indeed an important factor influencing its survival. Moreover, the distribution could be manipulated by inoculating soil at different moisture contents. Inoculation of dryer soils, as well as the use of higher inoculum densities, resulted in higher survival levels, which could be well explained with the concept of distribution of cells over protective and non-protective pore space. The occurance of different population levels under apparently the same environmental conditions during incubation, suggests that extensive translocation in natural soil is absent.

    The ability to manipulate the distribution and thereby to influence the survival of introduced bacteria, is important for the application of bacteria in soil. The availability of methods for biological control of soil-borne pathogens, nitrogen fixation and degradation of xenobiotics in soil, is depending on the possibility of introduced bacteria to establish in soil. The knowledge obtained in this research project can be used to improve the survival of bacteria introduced into soil.

    The spatial distribution of bacteria through the soil matrix might also be a useful concept for other areas in soil(micro)biology. The occurance, for example, of genetransfer in different soil systems might be better understood when more details about bacterial distribution are available. Also the preservation of organic matter and the activity of predators will depend on the distribution of bacteria in the soil matrix.

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