Publications by authors named "Wilfred F M Röling"

59 Publications

High biodiversity in a benzene-degrading nitrate-reducing culture is sustained by a few primary consumers.

Commun Biol 2021 May 5;4(1):530. Epub 2021 May 5.

Department of Molecular Cell Biology, AIMMS, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

A key question in microbial ecology is what the driving forces behind the persistence of large biodiversity in natural environments are. We studied a microbial community with more than 100 different types of species which evolved in a 15-years old bioreactor with benzene as the main carbon and energy source and nitrate as the electron acceptor. Using genome-centric metagenomics plus metatranscriptomics, we demonstrate that most of the community members likely feed on metabolic left-overs or on necromass while only a few of them, from families Rhodocyclaceae and Peptococcaceae, are candidates to degrade benzene. We verify with an additional succession experiment using metabolomics and metabarcoding that these few community members are the actual drivers of benzene degradation. As such, we hypothesize that high species richness is maintained and the complexity of a natural community is stabilized in a controlled environment by the interdependencies between the few benzene degraders and the rest of the community members, ultimately resulting in a food web with different trophic levels.
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http://dx.doi.org/10.1038/s42003-021-01948-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099898PMC
May 2021

Ample Arsenite Bio-Oxidation Activity in Bangladesh Drinking Water Wells: A Bonanza for Bioremediation?

Microorganisms 2019 Aug 8;7(8). Epub 2019 Aug 8.

Department of Molecular Cell Biology, Faculty of Science, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.

Millions of people worldwide are at risk of arsenic poisoning from their drinking water. In Bangladesh the problem extends to rural drinking water wells, where non-biological solutions are not feasible. In serial enrichment cultures of water from various Bangladesh drinking water wells, we found transfer-persistent arsenite oxidation activity under four conditions (aerobic/anaerobic; heterotrophic/autotrophic). This suggests that biological decontamination may help ameliorate the problem. The enriched microbial communities were phylogenetically at least as diverse as the unenriched communities: they contained a bonanza of 16S rRNA gene sequences. These related to , , , and species. In addition, the enriched microbiomes contained genes highly similar to the arsenite oxidase () gene of chemolithoautotrophic (e.g., sp. SY) and heterotrophic arsenite-oxidizing strains. The enriched cultures also contained phylotypes not detected in the previous survey of uncultivated samples from the same wells. Anaerobic enrichments disclosed a wider diversity of arsenite oxidizing phylotypes than did aerobic enrichments. The cultivatable chemolithoautotrophic and heterotrophic arsenite oxidizers are of great interest for future or arsenic bioremediation technologies for the detoxification of drinking water by oxidizing arsenite to arsenate that should then precipitates with iron oxides. The microbial activities required for such a technology seem present, amplifiable, diverse and hence robust.
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http://dx.doi.org/10.3390/microorganisms7080246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723331PMC
August 2019

Subsurface landfill leachate contamination affects microbial metabolic potential and gene expression in the Banisveld aquifer.

FEMS Microbiol Ecol 2018 10;94(10)

Molecular Cell Physiology, Vrije Universiteit Amsterdam, De Boelelaan 1085 HV Amsterdam, the Netherlands.

Microbial communities in groundwater ecosystems can develop the capacity to degrade complex mixtures of chemicals resulting from pollution by landfill leachate. Monitoring this natural attenuation requires insight into the metabolic potential and activity of microbial communities. We contrasted the metagenomes and metatranscriptomes from a leachate-polluted aquifer downstream of the Banisveld (the Netherlands) landfill with uncontaminated groundwater, which revealed changes in microbial genomic content and activity. Banisveld landfill leachate contains mono-aromatic hydrocarbons and the assessment of natural attenuation of these compounds in the aquifer had been a focal point of research. In the contaminated groundwater, active microbial functions were the ones involved in degradation of complex carbon substrates and organic pollutants. We found that benzylsuccinate synthase genes-involved in the catabolism of toluene-were highly expressed close to the source of contamination, confirming the ongoing natural attenuation of organic mono-aromatic hydrocarbon pollution in this aquifer. Additionally, metatranscriptomes were indicative of phosphorus limitation that can constrain total microbial activity and agree with the low phosphate concentrations (<0.4 μmol/L) in this aquifer. Through the application of metagenomics and metatranscriptomics, we were able to determine functional potential and expression patterns to assess the natural attenuation processes and constraints on microbial communities.
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http://dx.doi.org/10.1093/femsec/fiy156DOI Listing
October 2018

Metabolic flexibility of a prospective bioremediator: Desulfitobacterium hafniense Y51 challenged in chemostats.

Environ Microbiol 2018 07;20(7):2652-2669

Molecular Cell Physiology, Faculty of Science, VU University Amsterdam, De Boelelaan 1085, 1081, HV, Amsterdam, The Netherlands.

Desulfitobacterium hafniense Y51 has been widely used in investigations of perchloroethylene (PCE) biodegradation, but limited information exists on its other physiological capabilities. We investigated how D. hafniense Y51 confronts the debilitating limitations of not having enough electron donor (lactate), or electron acceptor (fumarate) during cultivation in chemostats. The residual concentrations of the substrates supplied in excess were much lower than expected. Transcriptomics, proteomics and fluxomics were integrated to investigate how this phenomenon was regulated. Through diverse regulation at both transcriptional and translational levels, strain Y51 turned to fermenting the excess lactate and disproportionating the excess fumarate under fumarate- and lactate-limiting conditions respectively. Genes and proteins related to the utilization of a variety of alternative electron donors and acceptors absent from the medium were induced, apparently involving the Wood-Ljungdahl pathway. Through this metabolic flexibility, D. hafniense Y51 may be able to switch between different metabolic capabilities under limiting conditions.
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http://dx.doi.org/10.1111/1462-2920.14295DOI Listing
July 2018

Formulation and stabilization of an Arthrobacter strain with good storage stability and 4-chlorophenol-degradation activity for bioremediation.

Appl Microbiol Biotechnol 2018 Feb 18;102(4):2031-2040. Epub 2018 Jan 18.

Department of Molecular Sciences, Swedish University of Agricultural Sciences-SLU, Uppsala, Sweden.

Chlorophenols are widespread and of environmental concern due to their toxic and carcinogenic properties. Development of less costly and less technically challenging remediation methods are needed; therefore, we developed a formulation based on micronized vermiculite that, when air-dried, resulted in a granular product containing the 4-chlorophenol (4-CP)-degrading Gram-positive bacterium Arthrobacter chlorophenolicus A6. This formulation and stabilization method yielded survival rates of about 60% that remained stable in storage for at least 3 months at 4 °C. The 4-CP degradation by the formulated and desiccated A. chlorophenolicus A6 cells was compared to that of freshly grown cells in controlled-environment soil microcosms. The stabilized cells degraded 4-CP equally efficient as freshly grown cells in two different set-ups using both hygienized and non-treated soils. The desiccated microbial product was successfully employed in an outdoor pot trial showing its effectiveness under more realistic environmental conditions. No significant phytoremediation effects on 4-CP degradation were observed in the outdoor pot experiment. The 4-CP degradation kinetics from both the microcosms and the outdoor pot trial were used to generate a predictive model of 4-CP biodegradation potentially useful for larger-scale operations, enabling better bioremediation set-ups and saving of resources. This study also opens up the possibility of formulating and stabilizing also other Arthrobacter strains possessing different desirable pollutant-degrading capabilities.
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http://dx.doi.org/10.1007/s00253-017-8706-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794804PMC
February 2018

Compositional Stability of the Bacterial Community in a Climate-Sensitive Sub-Arctic Peatland.

Front Microbiol 2017 7;8:317. Epub 2017 Mar 7.

Department of Ecological Science, Vrije Universiteit AmsterdamAmsterdam, Netherlands; Institute of Environmental Sciences, Leiden UniversityLeiden, Netherlands.

The climate sensitivity of microbe-mediated soil processes such as carbon and nitrogen cycling offers an interesting case for evaluating the corresponding sensitivity of microbial community composition to environmental change. Better understanding of the degree of linkage between functional and compositional stability would contribute to ongoing efforts to build mechanistic models aiming at predicting rates of microbe-mediated processes. We used an amplicon sequencing approach to test if previously observed large effects of experimental soil warming on C and N cycle fluxes (50-100% increases) in a sub-arctic peatland were reflected in changes in the composition of the soil bacterial community. We found that treatments that previously induced changes to fluxes did not associate with changes in the phylogenetic composition of the soil bacterial community. For both DNA- and RNA-based analyses, variation in bacterial communities could be explained by the hierarchy: spatial variation (12-15% of variance explained) > temporal variation (7-11%) > climate treatment (4-9%). We conclude that the bacterial community in this environment is stable under changing conditions, despite the previously observed sensitivity of process rates-evidence that microbe-mediated soil processes can alter without concomitant changes in bacterial communities. We propose that progress in linking soil microbial communities to ecosystem processes can be advanced by further investigating the relative importance of community composition effects versus physico-chemical factors in controlling biogeochemical process rates in different contexts.
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http://dx.doi.org/10.3389/fmicb.2017.00317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339224PMC
March 2017

Model-based quantification of metabolic interactions from dynamic microbial-community data.

PLoS One 2017 9;12(3):e0173183. Epub 2017 Mar 9.

Systems Bioinformatics, Amsterdam Insititute for Molecules, Medicines and Systems, VU Amsterdam, The Netherlands.

An important challenge in microbial ecology is to infer metabolic-exchange fluxes between growing microbial species from community-level data, concerning species abundances and metabolite concentrations. Here we apply a model-based approach to integrate such experimental data and thereby infer metabolic-exchange fluxes. We designed a synthetic anaerobic co-culture of Clostridium acetobutylicum and Wolinella succinogenes that interact via interspecies hydrogen transfer and applied different environmental conditions for which we expected the metabolic-exchange rates to change. We used stoichiometric models of the metabolism of the two microorganisms that represents our current physiological understanding and found that this understanding - the model - is sufficient to infer the identity and magnitude of the metabolic-exchange fluxes and it suggested unexpected interactions. Where the model could not fit all experimental data, it indicates specific requirement for further physiological studies. We show that the nitrogen source influences the rate of interspecies hydrogen transfer in the co-culture. Additionally, the model can predict the intracellular fluxes and optimal metabolic exchange rates, which can point to engineering strategies. This study therefore offers a realistic illustration of the strengths and weaknesses of model-based integration of heterogenous data that makes inference of metabolic-exchange fluxes possible from community-level experimental data.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173183PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5344373PMC
September 2017

Physiological and Transcriptome Response of the Polycyclic Aromatic Hydrocarbon Degrading Novosphingobium sp. LH128 after Inoculation in Soil.

Environ Sci Technol 2017 02 18;51(3):1570-1579. Epub 2017 Jan 18.

Division of Soil and Water Management, KU Leuven , Kasteelpark Arenberg 20, 3001 Heverlee, Belgium.

Soil bioaugmentation involves the inoculation of pollutant-degrading bacteria to accelerate pollutant degradation. Often the inoculum shows a dramatic decrease in Colony Forming Units (CFU) upon soil inoculation but this behavior is not well-understood. In this study, the physiology and transcriptomic response of a GFP tagged variant of Novosphingobium sp. LH128 was examined after inoculation into phenanthrene spiked soil. Four hours after inoculation, strain LH128-GFP showed about 99% reduction in CFU while microscopic counts of GFP-expressing cells were identical to the expected initial cell density, indicating that the reduction in CFU number is explained by cells entering into a Viable But Non-Culturable (VBNC)-like state and not by cell death. Transcriptome analysis showed a remarkably higher expression of phenanthrene degradation genes 4 h after inoculation, compared to the inoculum suspension concomitant with an increased expression of genes involved in stress response. This indicates that the cells were active in phenanthrene degradation while experiencing stress. Between 4 h and 10 days, CFU numbers increased to numbers comparable to the inoculated cell density. Our results suggest that strain LH128-GFP enters a VBNC-like state upon inoculation into soil but is metabolically active and that VBNC cells should be taken into account in evaluating bioaugmentation approaches.
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http://dx.doi.org/10.1021/acs.est.6b03822DOI Listing
February 2017

Resilience of Soil Microbial Communities to Metals and Additional Stressors: DNA-Based Approaches for Assessing "Stress-on-Stress" Responses.

Int J Mol Sci 2016 Jun 14;17(6). Epub 2016 Jun 14.

Department of Ecological Science, Faculty of Earth and Life Sciences, Vrije Universiteit, de Boelelaan 1085, 1081HV Amsterdam, The Netherlands.

Many microbial ecology studies have demonstrated profound changes in community composition caused by environmental pollution, as well as adaptation processes allowing survival of microbes in polluted ecosystems. Soil microbial communities in polluted areas with a long-term history of contamination have been shown to maintain their function by developing metal-tolerance mechanisms. In the present work, we review recent experiments, with specific emphasis on studies that have been conducted in polluted areas with a long-term history of contamination that also applied DNA-based approaches. We evaluate how the "costs" of adaptation to metals affect the responses of metal-tolerant communities to other stress factors ("stress-on-stress"). We discuss recent studies on the stability of microbial communities, in terms of resistance and resilience to additional stressors, focusing on metal pollution as the initial stress, and discuss possible factors influencing the functional and structural stability of microbial communities towards secondary stressors. There is increasing evidence that the history of environmental conditions and disturbance regimes play central roles in responses of microbial communities towards secondary stressors.
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http://dx.doi.org/10.3390/ijms17060933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926466PMC
June 2016

The microbiome of Folsomia candida: an assessment of bacterial diversity in a Wolbachia-containing animal.

FEMS Microbiol Ecol 2015 Nov 22;91(11). Epub 2015 Oct 22.

Department of Molecular Cell Physiology, VU University Amsterdam, De Boelelaan 1085-1087, 1081 HV Amsterdam, the Netherlands.

The springtail Folsomia candida is an important model organism for soil ecology, ecotoxicology and ecogenomics. The decomposer activities of soil invertebrates like Folsomia depend on their relationship with microbial communities including gut symbionts. In this paper, we apply high-throughput sequencing to provide a detailed characterization of the bacterial community associated with parthenogenetic F. candida. First, we evaluated a method to suppress the amplification of DNA from the endosymbiont Wolbachia, to prevent it from interfering with the identification of less abundant operational taxonomic units (OTUs). The suppression treatment applied was effective against Wolbachia and did not interfere with the detection of the most abundant OTUs (59 OTUs, contributing over 87% of the reads). However, this method did affect the inferred community composition. Significant differences were subsequently observed in the composition of bacterial communities associated with two different strains of F. candida. A total of 832 OTUs were found, of which 45% were only present in one strain and 17% only in the other. Among the 20 most abundant OTUs, 16 were shared between strains. Denaturing gradient gel electrophoresis and clone libraries, although unable to capture the full diversity of the bacterial community, provided results that supported the NGS data.
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http://dx.doi.org/10.1093/femsec/fiv128DOI Listing
November 2015

The Significance of Microbe-Mineral-Biomarker Interactions in the Detection of Life on Mars and Beyond.

Astrobiology 2015 Jun;15(6):492-507

1 Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam , Amsterdam, the Netherlands .

The detection of biomarkers plays a central role in our effort to establish whether there is, or was, life beyond Earth. In this review, we address the importance of considering mineralogy in relation to the selection of locations and biomarker detection methodologies with characteristics most promising for exploration. We review relevant mineral-biomarker and mineral-microbe interactions. The local mineralogy on a particular planet reflects its past and current environmental conditions and allows a habitability assessment by comparison with life under extreme conditions on Earth. The type of mineral significantly influences the potential abundances and types of biomarkers and microorganisms containing these biomarkers. The strong adsorptive power of some minerals aids in the preservation of biomarkers and may have been important in the origin of life. On the other hand, this strong adsorption as well as oxidizing properties of minerals can interfere with efficient extraction and detection of biomarkers. Differences in mechanisms of adsorption and in properties of minerals and biomarkers suggest that it will be difficult to design a single extraction procedure for a wide range of biomarkers. While on Mars samples can be used for direct detection of biomarkers such as nucleic acids, amino acids, and lipids, on other planetary bodies remote spectrometric detection of biosignatures has to be relied upon. The interpretation of spectral signatures of photosynthesis can also be affected by local mineralogy. We identify current gaps in our knowledge and indicate how they may be filled to improve the chances of detecting biomarkers on Mars and beyond.
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http://dx.doi.org/10.1089/ast.2014.1276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490593PMC
June 2015

Systems modeling approaches for microbial community studies: from metagenomics to inference of the community structure.

Front Microbiol 2015 19;6:213. Epub 2015 Mar 19.

Systems Bioinformatics, Amsterdam Institute for Molecules, Medicines and Systems, VU University Amsterdam Amsterdam, Netherlands.

Microbial communities play important roles in health, industrial applications and earth's ecosystems. With current molecular techniques we can characterize these systems in unprecedented detail. However, such methods provide little mechanistic insight into how the genetic properties and the dynamic couplings between individual microorganisms give rise to their dynamic activities. Neither do they give insight into what we call "the community state", that is the fluxes and concentrations of nutrients within the community. This knowledge is a prerequisite for rational control and intervention in microbial communities. Therefore, the inference of the community structure from experimental data is a major current challenge. We will argue that this inference problem requires mathematical models that can integrate heterogeneous experimental data with existing knowledge. We propose that two types of models are needed. Firstly, mathematical models that integrate existing genomic, physiological, and physicochemical information with metagenomics data so as to maximize information content and predictive power. This can be achieved with the use of constraint-based genome-scale stoichiometric modeling of community metabolism which is ideally suited for this purpose. Next, we propose a simpler coarse-grained model, which is tailored to solve the inference problem from the experimental data. This model unambiguously relate to the more detailed genome-scale stoichiometric models which act as heterogeneous data integrators. The simpler inference models are, in our opinion, key to understanding microbial ecosystems, yet until now, have received remarkably little attention. This has led to the situation where the modeling of microbial communities, using only genome-scale models is currently more a computational, theoretical exercise than a method useful to the experimentalist.
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http://dx.doi.org/10.3389/fmicb.2015.00213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365725PMC
April 2015

Diverse arsenic- and iron-cycling microbial communities in arsenic-contaminated aquifers used for drinking water in Bangladesh.

FEMS Microbiol Ecol 2015 Apr 15;91(4). Epub 2015 Mar 15.

Department of Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam, 1081 HV Amsterdam, the Netherlands

Subsurface removal of arsenic by injection with oxygenated groundwater has been proposed as a viable technology for obtaining 'safe' drinking water in Bangladesh. While the oxidation of ferrous iron to solid ferric iron minerals, to which arsenic adsorbs, is assumed to be driven by abiotic reactions, metal-cycling microorganisms may potentially affect arsenic removal. A cultivation-independent survey covering 24 drinking water wells in several geographical regions in Bangladesh was conducted to obtain information on microbial community structure and diversity in general, and on specific functional groups capable of the oxidation or reduction of arsenic or iron. Each functional group, targeted by either group-specific 16S rRNA or functional gene amplification, occurred in at least 79% of investigated samples. Putative arsenate reducers and iron-oxidizing Gallionellaceae were present at low diversity, while more variation in potentially arsenite-oxidizing microorganisms and iron-reducing Desulfuromonadales was revealed within and between samples. Relations between community composition on the one hand and hydrochemistry on the other hand were in general not evident, apart from an impact of salinity on iron-cycling microorganisms. Our data suggest widespread potential for a positive contribution of arsenite and iron oxidizers to arsenic removal upon injection with oxygenated water, but also indicate a potential risk for arsenic re-mobilization by anaerobic arsenate and iron reducers once injection is halted.
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http://dx.doi.org/10.1093/femsec/fiv026DOI Listing
April 2015

Microbial community composition and functions are resilient to metal pollution along two forest soil gradients.

FEMS Microbiol Ecol 2015 Jan 5;91(1):1-11. Epub 2014 Dec 5.

Department of Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.

Despite the global importance of forests, it is virtually unknown how their soil microbial communities adapt at the phylogenetic and functional level to long-term metal pollution. Studying 12 sites located along two distinct gradients of metal pollution in Southern Poland revealed that functional potential and diversity (assessed using GeoChip 4.2) were highly similar across the gradients despite drastically diverging metal contamination levels. Metal pollution level did, however, significantly impact bacterial community structure (as shown by MiSeq Illumina sequencing of 16S rRNA genes), but not bacterial taxon richness and community composition. Metal pollution caused changes in the relative abundance of specific bacterial taxa, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes and Proteobacteria. Also, a group of metal-resistance genes showed significant correlations with metal concentrations in soil. Our study showed that microbial communities are resilient to metal pollution; despite differences in community structure, no clear impact of metal pollution levels on overall functional diversity was observed. While screens of phylogenetic marker genes, such as 16S rRNA genes, provide only limited insight into resilience mechanisms, analysis of specific functional genes, e.g. involved in metal resistance, appears to be a more promising strategy.
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http://dx.doi.org/10.1093/femsec/fiu003DOI Listing
January 2015

Maths on microbes: adding microbial ecophysiology to metagenomics.

Microb Biotechnol 2015 Jan 9;8(1):21-2. Epub 2014 Dec 9.

Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam, Boelelaan 1085, 1081 HV, Amsterdam, the Netherlands.

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http://dx.doi.org/10.1111/1751-7915.12233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321363PMC
January 2015

Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation.

Microb Ecol 2015 Feb 30;69(2):422-33. Epub 2014 Nov 30.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam, Gustav Mahlerlaan 3004, 1081 LA, Amsterdam, The Netherlands,

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.
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http://dx.doi.org/10.1007/s00248-014-0535-xDOI Listing
February 2015

Biota and biomolecules in extreme environments on Earth: implications for life detection on Mars.

Life (Basel) 2014 Oct 13;4(4):535-65. Epub 2014 Oct 13.

Leiden Observatory, Leiden University, P.O. Box 9513, NL-2300 RA Leiden, The Netherlands.

The three main requirements for life as we know it are the presence of organic compounds, liquid water, and free energy. Several groups of organic compounds (e.g., amino acids, nucleobases, lipids) occur in all life forms on Earth and are used as diagnostic molecules, i.e., biomarkers, for the characterization of extant or extinct life. Due to their indispensability for life on Earth, these biomarkers are also prime targets in the search for life on Mars. Biomarkers degrade over time; in situ environmental conditions influence the preservation of those molecules. Nonetheless, upon shielding (e.g., by mineral surfaces), particular biomarkers can persist for billions of years, making them of vital importance in answering questions about the origins and limits of life on early Earth and Mars. The search for organic material and biosignatures on Mars is particularly challenging due to the hostile environment and its effect on organic compounds near the surface. In support of life detection on Mars, it is crucial to investigate analogue environments on Earth that resemble best past and present Mars conditions. Terrestrial extreme environments offer a rich source of information allowing us to determine how extreme conditions affect life and molecules associated with it. Extremophilic organisms have adapted to the most stunning conditions on Earth in environments with often unique geological and chemical features. One challenge in detecting biomarkers is to optimize extraction, since organic molecules can be low in abundance and can strongly adsorb to mineral surfaces. Methods and analytical tools in the field of life science are continuously improving. Amplification methods are very useful for the detection of low concentrations of genomic material but most other organic molecules are not prone to amplification methods. Therefore, a great deal depends on the extraction efficiency. The questions "what to look for", "where to look", and "how to look for it" require more of our attention to ensure the success of future life detection missions on Mars.
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http://dx.doi.org/10.3390/life4040535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284457PMC
October 2014

Physiology of Geobacter metallireducens under excess and limitation of electron donors. Part II. Mimicking environmental conditions during cultivation in retentostats.

Syst Appl Microbiol 2014 Jun 13;37(4):287-95. Epub 2014 Apr 13.

Institute of Groundwater Ecology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany. Electronic address:

The strict anaerobe Geobacter metallireducens was cultivated in retentostats under acetate and acetate plus benzoate limitation in the presence of Fe(III) citrate in order to investigate its physiology under close to natural conditions. Growth rates below 0.003h(-1) were achieved in the course of cultivation. A nano-liquid chromatography-tandem mass spectrometry-based proteomic approach (nano-LC-MS/MS) with subsequent label-free quantification was performed on proteins extracted from cells sampled at different time points during retentostat cultivation. Proteins detected at low (0.002h(-1)) and high (0.06h(-1)) growth rates were compared between corresponding growth conditions (acetate or acetate plus benzoate). Carbon limitation significantly increased the abundances of several catabolic proteins involved in the degradation of substrates not present in the medium (ethanol, butyrate, fatty acids, and aromatic compounds). Growth rate-specific physiology was reflected in the changed abundances of energy-, chemotaxis-, oxidative stress-, and transport-related proteins. Mimicking natural conditions by extremely slow bacterial growth allowed to show how G. metallireducens optimized its physiology in order to survive in its natural habitats, since it was prepared to consume several carbon sources simultaneously and to withstand various environmental stresses.
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http://dx.doi.org/10.1016/j.syapm.2014.02.005DOI Listing
June 2014

Physiology of Geobacter metallireducens under excess and limitation of electron donors. Part I. Batch cultivation with excess of carbon sources.

Syst Appl Microbiol 2014 Jun 14;37(4):277-86. Epub 2014 Apr 14.

Institute of Groundwater Ecology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany. Electronic address:

For microorganisms that play an important role in bioremediation, the adaptation to swift changes in the availability of various substrates is a key for survival. The iron-reducing bacterium Geobacter metallireducens was hypothesized to repress utilization of less preferred substrates in the presence of high concentrations of easily degradable compounds. In our experiments, acetate and ethanol were preferred over benzoate, but benzoate was co-consumed with toluene and butyrate. To reveal overall physiological changes caused by different single substrates and a mixture of acetate plus benzoate, a nano-liquid chromatography-tandem mass spectrometry-based proteomic approach (nano-LC-MS/MS) was performed using label-free quantification. Significant differential expression during growth on different substrates was observed for 155 out of 1477 proteins. The benzoyl-CoA pathway was found to be subjected to incomplete repression during exponential growth on acetate in the presence of benzoate and on butyrate as a single substrate. Peripheral pathways of toluene, ethanol, and butyrate degradation were highly expressed only during growth on the corresponding substrates. However, low expression of these pathways was detected in all other tested conditions. Therefore, G. metallireducens seems to lack strong carbon catabolite repression under high substrate concentrations, which might be advantageous for survival in habitats rich in fatty acids and aromatic hydrocarbons.
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http://dx.doi.org/10.1016/j.syapm.2014.02.004DOI Listing
June 2014

Toward quantitative understanding on microbial community structure and functioning: a modeling-centered approach using degradation of marine oil spills as example.

Front Microbiol 2014 26;5:125. Epub 2014 Mar 26.

Systems Ecology, Department of Ecological Sciences, Faculty of Earth and Life Sciences, VU University Amsterdam Amsterdam, Netherlands.

Molecular ecology approaches are rapidly advancing our insights into the microorganisms involved in the degradation of marine oil spills and their metabolic potentials. Yet, many questions remain open: how do oil-degrading microbial communities assemble in terms of functional diversity, species abundances and organization and what are the drivers? How do the functional properties of microorganisms scale to processes at the ecosystem level? How does mass flow among species, and which factors and species control and regulate fluxes, stability and other ecosystem functions? Can generic rules on oil-degradation be derived, and what drivers underlie these rules? How can we engineer oil-degrading microbial communities such that toxic polycyclic aromatic hydrocarbons are degraded faster? These types of questions apply to the field of microbial ecology in general. We outline how recent advances in single-species systems biology might be extended to help answer these questions. We argue that bottom-up mechanistic modeling allows deciphering the respective roles and interactions among microorganisms. In particular constraint-based, metagenome-derived community-scale flux balance analysis appears suited for this goal as it allows calculating degradation-related fluxes based on physiological constraints and growth strategies, without needing detailed kinetic information. We subsequently discuss what is required to make these approaches successful, and identify a need to better understand microbial physiology in order to advance microbial ecology. We advocate the development of databases containing microbial physiological data. Answering the posed questions is far from trivial. Oil-degrading communities are, however, an attractive setting to start testing systems biology-derived models and hypotheses as they are relatively simple in diversity and key activities, with several key players being isolated and a high availability of experimental data and approaches.
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http://dx.doi.org/10.3389/fmicb.2014.00125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972468PMC
April 2014

Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

Environ Microbiol 2014 Mar 23;16(3):643-57. Epub 2014 Jan 23.

Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam, Amsterdam, The Netherlands.

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars.
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http://dx.doi.org/10.1111/1462-2920.12365DOI Listing
March 2014

Interactions between accumulated copper, bacterial community structure and histamine levels in crayfish meat during storage.

J Sci Food Agric 2014 Aug 15;94(10):2023-9. Epub 2014 Jan 15.

Department of Ecological Science, VU University Amsterdam, 1081, HV, Amsterdam, the Netherlands; Department of Food Technology, Faculty of Agricultural Technology, Soegijapranata Catholic University, Semarang, 50234, Central Java, Indonesia.

Background: Pollution in aquaculture areas may negatively impact edible species and threaten seafood quality and safety. The aim of this study was to determine the interaction between copper and bacteria in the aquatic habitat and their impact upon crustaceans. Marbled crayfish was chosen as a model of aquatic crustaceans and the influence of metal contamination on bacterial community structure in water used to culture crayfish and in crayfish themselves was investigated. Histamine, an allergen commonly formed by certain groups of bacteria in crustacean edible tissue during storage, was also determined.

Results: Copper exposure increased its concentration in crayfish meat by 17.4%, but the copper concentration remained within acceptable food safety limits. Elevated copper levels affected the bacterial community both in the water used to cultivate crayfish and in the marbled crayfish themselves. Cluster analysis of 16S rRNA-gene based microbial community fingerprints revealed that copper impacted the bacterial community in the water and in the crayfish meat. However, copper exposure reduced the formation of histamine in crayfish meat during storage by 66.3%.

Conclusion: Copper from the habitat appears to reduce histamine accumulation in crayfish meat during storage by affecting the bacterial community structure of the cultivation water and most likely also in the intestine of the crayfish. From a food safety point of view, copper treatment during the aqua culturing of crustaceans has a positive impact on the postharvest stage.
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http://dx.doi.org/10.1002/jsfa.6519DOI Listing
August 2014

Impacts of shallow geothermal energy production on redox processes and microbial communities.

Environ Sci Technol 2013 Dec 4;47(24):14476-84. Epub 2013 Dec 4.

KWR Watercycle Research Institute , P.O. Box 1072, 3430BB Nieuwegein, The Netherlands .

Shallow geothermal systems are increasingly being used to store or harvest thermal energy for heating or cooling purposes. This technology causes temperature perturbations exceeding the natural variations in aquifers, which may impact groundwater quality. Here, we report the results of laboratory experiments on the effect of temperature variations (5-80 °C) on redox processes and associated microbial communities in anoxic unconsolidated subsurface sediments. Both hydrochemical and microbiological data showed that a temperature increase from 11 °C (in situ) to 25 °C caused a shift from iron-reducing to sulfate-reducing and methanogenic conditions. Bioenergetic calculations could explain this shift. A further temperature increase (>45 °C) resulted in the emergence of a thermophilic microbial community specialized in fermentation and sulfate reduction. Two distinct maxima in sulfate reduction rates, of similar orders of magnitude (5 × 10(-10) M s(-1)), were observed at 40 and 70 °C. Thermophilic sulfate reduction, however, had a higher activation energy (100-160 kJ mol(-1)) than mesophilic sulfate reduction (30-60 kJ mol(-1)), which might be due to a trade-off between enzyme stability and activity with thermostable enzymes being less efficient catalysts that require higher activation energies. These results reveal that while sulfate-reducing functionality can withstand a substantial temperature rise, other key biochemical processes appear more temperature sensitive.
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http://dx.doi.org/10.1021/es4030244DOI Listing
December 2013

Tracking fungal community responses to maize plants by DNA- and RNA-based pyrosequencing.

PLoS One 2013 18;8(7):e69973. Epub 2013 Jul 18.

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands.

We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age) in pots associated with four maize cultivars, including two genetically modified (GM) cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA). The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most "active" fungi (as recovered via RNA). Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production). Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069973PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715498PMC
February 2014

Community flux balance analysis for microbial consortia at balanced growth.

PLoS One 2013 31;8(5):e64567. Epub 2013 May 31.

Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam, Amsterdam, The Netherlands.

A central focus in studies of microbial communities is the elucidation of the relationships between genotype, phenotype, and dynamic community structure. Here, we present a new computational method called community flux balance analysis (cFBA) to study the metabolic behavior of microbial communities. cFBA integrates the comprehensive metabolic capacities of individual microorganisms in terms of (genome-scale) stoichiometric models of metabolism, and the metabolic interactions between species in the community and abiotic processes. In addition, cFBA considers constraints deriving from reaction stoichiometry, reaction thermodynamics, and the ecosystem. cFBA predicts for communities at balanced growth the maximal community growth rate, the required rates of metabolic reactions within and between microbes and the relative species abundances. In order to predict species abundances and metabolic activities at the optimal community growth rate, a nonlinear optimization problem needs to be solved. We outline the methodology of cFBA and illustrate the approach with two examples of microbial communities. These examples illustrate two useful applications of cFBA. Firstly, cFBA can be used to study how specific biochemical limitations in reaction capacities cause different types of metabolic limitations that microbial consortia can encounter. In silico variations of those maximal capacities allow for a global view of the consortium responses to various metabolic and environmental constraints. Secondly, cFBA is very useful for comparing the performance of different metabolic cross-feeding strategies to either find one that agrees with experimental data or one that is most efficient for the community of microorganisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064567PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3669319PMC
February 2014

Microbial community structure and functioning along metal pollution gradients.

Environ Toxicol Chem 2013 Sep 8;32(9):1992-2002. Epub 2013 Jul 8.

Institute of Environmental Sciences, Jagiellonian University, Krakow, Poland.

Toxic effects of heavy metals on soil microorganisms have been confirmed in a number of laboratory studies. However, most real-field studies do not allow for strong general conclusions due to a range of problems, such as pseudoreplication and confounding factors, which are almost impossible to control for with the most commonly used polluted versus unpolluted or random sampling designs. Effects of metal contamination on soil microbial community traits were measured along 2 pollution gradients in southern Poland. Employing an experimental regression design, using 2 separate gradients, the authors aimed to control for effects of soil properties and beta-diversity of microbial communities. General microbial activity was measured as soil basal respiration rate and substrate-induced respiration, while microbial functional and structural diversity were analyzed with community-level physiological profiles and phospholipid fatty acid patterns, respectively. Metal concentrations were normalized to their toxicity and integrated in a toxicity index (TI). Microbial activity (basal and substrate-induced respiration) decreased in both gradients with increasing TI. Community-level physiological profiles for fungi correlated positively with TI, but no impact of TI on the community-level physiological profiles of bacteria was observed. The phospholipid fatty acids a:15 and i:17 were positively correlated with TI, whereas 16:1ω9 and 18:2ω9 were negatively correlated with TI. The use of 2 gradients (Olkusz and Miasteczko Śląskie) allowed the authors to reveal a clear effect of pollution on general microbial structure and activities, even though they were not able to control completely for all confounding factors. Soil pH, organic matter content, and nutrient level appeared to be at least as important as TI in determining microbial community structure and activities.
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http://dx.doi.org/10.1002/etc.2269DOI Listing
September 2013

Testing potential effects of maize expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) on mycorrhizal fungal communities via DNA- and RNA-based pyrosequencing and molecular fingerprinting.

Appl Environ Microbiol 2012 Oct 10;78(20):7384-92. Epub 2012 Aug 10.

Department of Ecological Science, Faculty of Earth and Life Sciences, VU University, De Boelelaan, Amsterdam, The Netherlands.

The cultivation of genetically modified (GM) crops has increased significantly over the last decades. However, concerns have been raised that some GM traits may negatively affect beneficial soil biota, such as arbuscular mycorrhizal fungi (AMF), potentially leading to alterations in soil functioning. Here, we test two maize varieties expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) for their effects on soil AM fungal communities. We target both fungal DNA and RNA, which is new for AM fungi, and we use two strategies as an inclusive and robust way of detecting community differences: (i) 454 pyrosequencing using general fungal rRNA gene-directed primers and (ii) terminal restriction fragment length polymorphism (T-RFLP) profiling using AM fungus-specific markers. Potential GM-induced effects were compared to the normal natural variation of AM fungal communities across 15 different agricultural fields. AM fungi were found to be abundant in the experiment, accounting for 8% and 21% of total recovered DNA- and RNA-derived fungal sequences, respectively, after 104 days of plant growth. RNA- and DNA-based sequence analyses yielded most of the same AM fungal lineages. Our research yielded three major conclusions. First, no consistent differences were detected between AM fungal communities associated with GM plants and non-GM plants. Second, temporal variation in AMF community composition (between two measured time points) was bigger than GM trait-induced variation. Third, natural variation of AMF communities across 15 agricultural fields in The Netherlands, as well as within-field temporal variation, was much higher than GM-induced variation. In conclusion, we found no indication that Bt maize cultivation poses a risk for AMF.
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http://dx.doi.org/10.1128/AEM.01372-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457118PMC
October 2012

Community assembly, species richness and nestedness of arbuscular mycorrhizal fungi in agricultural soils.

Mol Ecol 2012 May 22;21(10):2341-53. Epub 2012 Mar 22.

Department of Ecological Science, Faculty of Earth of Life Sciences, VU University Amsterdam, the Netherlands.

Understanding how communities assemble is a central goal of ecology. This is particularly relevant for communities of arbuscular mycorrhizal fungi (AMF), because the community composition of these beneficial plant symbionts influences important ecosystem processes. Moreover, AMF may be used as sensitive indicators of ecological soil quality if they respond to environmental variation in a predictable way. Here, we use a molecular profiling technique (T-RFLP of 25S rRNA gene fragments) to test which factors determine AM fungal community composition in 40 agricultural soils in the Netherlands. In particular, we test whether species richness, dominance structure and community nestedness are influenced by management type (in pairs of organically and conventionally farmed fields), and we examine the contribution of crop species (maize vs. potato), soil type (sand vs. clay-textured soils) and habitat (plant root vs. bulk soil) on AMF community characteristics. AMF richness varied from 1 to 11 taxa per field. Communities from species-poor fields were found to be subsets of those in richer fields, indicating nestedness and a progressive 'loss' from the species pool. AMF taxa richness and occurrence in soil and plant roots were highly correlated, and richness was related to management intensity (phosphate availability and grass-cropping history together explained 32% and 50% of richness in roots and soils). Soil type together with soil chemical parameters explained only 17% of variance in AMF community structure. We synthesize these results by discussing the potential contribution of a 'bottleneck effect' on AMF communities through increased stochastic effects under environmental stress.
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http://dx.doi.org/10.1111/j.1365-294X.2012.05534.xDOI Listing
May 2012

Sensitive life detection strategies for low-biomass environments: optimizing extraction of nucleic acids adsorbing to terrestrial and Mars analogue minerals.

FEMS Microbiol Ecol 2012 Jul 7;81(1):111-23. Epub 2012 Mar 7.

Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam, Amsterdam, The Netherlands.

The adsorption of nucleic acids to mineral matrixes can result in low extraction yields and negatively influences molecular microbial ecology studies, in particular for low-biomass environments on Earth and Mars. We determined the recovery of nucleic acids from a range of minerals relevant to Earth and Mars. Clay minerals, but also other silicates and nonsilicates, showed very low recovery (< 1%). Consequently, optimization of DNA extraction was directed towards clays. The high temperatures and acidic conditions used in some methods to dissolve mineral matrices proved to destruct DNA. The most efficient method comprised a high phosphate solution (P/EtOH; 1 M phosphate, 15% ethanol buffer at pH 8) introduced at the cell-lysing step in DNA extraction, to promote chemical competition with DNA for adsorption sites. This solution increased DNA yield from clay samples spiked with known quantities of cells up to nearly 100-fold. DNA recovery was also enhanced from several mineral samples retrieved from an aquifer, while maintaining reproducible DGGE profiles. DGGE profiles were obtained for a clay sample for which no profile could be generated with the standard DNA isolation protocol. Mineralogy influenced microbial community composition. The method also proved suitable for the recovery of low molecular weight DNA (< 1.5 kb).
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http://dx.doi.org/10.1111/j.1574-6941.2012.01325.xDOI Listing
July 2012

The influence of long-term copper contaminated agricultural soil at different pH levels on microbial communities and springtail transcriptional regulation.

Environ Sci Technol 2012 Jan 16;46(1):60-8. Epub 2011 Sep 16.

Department of Ecological Sciences, VU University, de Boelelaan 1085, 1081HV, Amsterdam, The Netherlands.

Copper has long been applied for agricultural practises. Like other metals, copper is highly persistent in the environment and biologically active long after its use has ceased. Here we present a unique study on the long-term effects (27 years) of copper and pH on soil microbial communities and on the springtail Folsomia candida an important representative of the soil macrofauna, in an experiment with a full factorial, random block design. Bacterial communities were mostly affected by pH. These effects were prominent in Acidobacteria, while Actinobacteria and Gammaroteobacteria communities were affected by original and bioavailable copper. Reproduction and survival of the collembolan F. candida was not affected by the studied copper concentrations. However, the transcriptomic responses to copper reflected a mechanism of copper transport and detoxification, while pH exerted effects on nucleotide and protein metabolism and (acute) inflammatory response. We conclude that microbial community structure reflected the history of copper contamination, while gene expression analysis of F. candida is associated with the current level of bioavailable copper. The study is a first step in the development of a molecular strategy aiming at a more comprehensive assessment of various aspects of soil quality and ecotoxicology.
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http://dx.doi.org/10.1021/es2013598DOI Listing
January 2012