Subseafloor microbial communities associated with rapid turbidite deposition in the Gulf of Mexico continental slope (IODP Expedition 308)

The subseafloor microbial communities in the turbidite depositional basins Brazos-Trinity Basin IV (BT Basin) and the Mars-Ursa Basin (Ursa Basin) on the Gulf of Mexico continental slope (IODP holes U1319A, U1320A, U1322B and U1324B) were investigated by PCR-dependent molecular analyses targeted to the small subunit (SSU) rRNA genes, dsrA and mcrA , and hydrogenase activity measurements. Biomass at both basins was very low, with the maximum cell or the SSU rRNA gene copy number <1 × 10⁷ cells mL⁻¹ or copies g⁻¹ sediments, respectively. Hydrogenase activity correlated with biomass estimated by SSU rRNA gene copy number when all data sets were combined. We detected differences in the SSU rRNA gene community structures and SSU rRNA gene copy numbers between the basin-fill and basement sediments in the BT Basin. Examination of microbial communities and hydrogenase activity in the context of geochemical and geophysical parameters and sediment depositional environments revealed that differences in microbial community composition between the basin-fill and basement sediments in the BT Basin were associated with sedimentation regimes tied to the sea-level change. This may also explain the distributions of relatively similar archaeal communities in the Ursa Basin sediments and basement sediments in the BT Basin.     

The effect of lubricant contamination on ThinPrep® (Cytyc) cervical cytology liquid-based preparations

Objectives:  To assess the extent of lubricant use by smear-takers and the effect of lubricant contamination of ThinPrep^® processed cervical cytology samples.
Methods:  All primary care smear-takers were sent a questionnaire on lubricant type and frequency of use. Fifty cervical cytology samples were then contaminated with incremental amounts of K-Y^® jelly, 50 samples contaminated with incremental amounts of Aquagel^® and ten non-contaminated vials were processed using the ThinPrep^® T2000 processor followed by Papanicolaou staining. The morphological appearances of lubricant contamination were described microscopically and formal cell counts performed on all slides.
Results:  Seventy of 94 (74.5%) primary care smear-takers indicated lubricant use of whom 9/70 (12.8%) used Aquagel^® and 61/70 (87.2%) used K-Y^® jelly. K-Y^® jelly appeared as mucoid blue deposits in the slide background whereas Aquagel^® appeared as pink stringy background material. Cell counting showed a significant difference between Aquagel^® and K-Y^® jelly contaminated slides compared to the original non-contaminated preparations for all fields and the average fields ( P  < 0.001) with a significantly higher count for the original non-contaminated slides than the lubricant contaminated groups.
Conclusion:  Lubricant contamination of ThinPrep^® cervical cytology samples may result in reduced cellularity of the subsequent slide. This study provides evidence-based data to support British Society for Clinical Cytology recommendations for no lubricant use when taking cervical samples.     

Resistance and resilience of Cu-polluted soil after Cu perturbation, tested by a wide range of soil microbial parameters

Copper (Cu)-polluted and unpolluted soils were used to study the effect of initial pollution on soil biological resistance and resilience by measuring the responses to perturbation using different parameters. Microbial biomass carbon, substrate-induced respiration and copy numbers of 16S rRNA gene were grouped as general parameters, while potential ammonia oxidation rate and copy numbers of amo A gene were grouped as specific functions. In addition, to illustrate how initial pollution affects soil biological resistance and resilience following secondary perturbation, the microbial community structure, together with free Cu²⁺ activities ([Cu²⁺]) in soil pore water and soil pH were also measured after secondary perturbation. Results showed that general parameters were more stable than specific ones. High [Cu²⁺] and low pH in soil pore water induced by Cu addition may lead to apparently low resistance and resilience, whereas the formation of a tolerant community after Cu pollution, secondary perturbation and Cu aging may contribute to resistance and resilience. Analysis of the phospholipid fatty acids profile showed that microbial community structure shifted along with the [Cu²⁺] gradient. The microbial community structure of the control soil was both resistant and resilient to 400 mg kg⁻¹ Cu perturbation, whereas other treatments were neither resistant nor resilient.     

Taxon-specific associations between protozoal and methanogen populations in the rumen and a model rumen system

Methanogen populations in the rumen and in model rumen systems (operated over a 240-h period) were studied using the small subunit (SSU) rRNA phylogenetic framework for group-specific enumerations. Representatives of the family Methanobacteriaceae were the most abundant methanogen population in the rumen, accounting for 89.3% (± 1.02%) of total archaea in the rumen fluid and 99.2% (± 1.8%) in a protozoal fraction of rumen fluid. Their percentage of archaea in the model rumen systems declined from 84% (± 8.5%) to 54% (± 7.8%) after 48 h of operation, correlated with loss of protozoa from these systems. The Methanomicrobiales, encompassed by the families Methanomicrobiaceae, Methanocorpusculaceae, and Methanospirillaceae were the second most abundant population and accounted for 12.1% (± 2.15%) of total SSU rRNA in rumen fluid. Additionally this group was shown to be essentially free living, since only a negligible hybridization signal was detected with the ruminal protozoal fraction. This group constituted a more significant proportion of total archaea in whole rumen fluid, 12.1% (± 2.1%) and model rumen fluid containing no protozoa (26.3 ± 7.7%). In contrast, the Methanosarcinales, generally considered the second most abundant population of rumen methanogens, accounted for only 2.8% (± 0.3%) of total archaeal SSU rRNA in rumen fluid.     

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

RAPID ASSESSMENT OF POULTRY FEED MICROBIAL QUALITY USING POLYMERASE CHAIN REACTION DETECTION OF MICROBIAL RIBOSOMAL GENE SEQUENCES

Rapid assessment of microbial quality in poultry feeds is critical for detection of specific pathogens and to directly evaluate the effectiveness of potential antimicrobial compounds. This requires methodology that is not only rapid and sensitive but also representative of the diverse microflora present on poultry feeds. The objective of this study was to examine a PCR-based assay using conserved 16S rRNA ribosomal sequences to evaluate microbial quality in naturally contaminated poultry feed samples. When examined on gel electrophoresis, PCR assays yielded visible bands detectable in at least 1 replicate from 6 out of 8 poultry feed samples. Further methodology improvement may yield a consistent method for rapid determination of feed microbial diversity and feed quality assessment as a function of specific feed and feed ingredient microbial profiles.     

Impact of two bacterial biocontrol agents on bacterial and fungal culturable groups associated with the roots of field-grown maize

Aims:  To assess the impact of Bacillus amyloliquefaciens and Microbacterium oleovorans on bacterial and fungal groups associated to the roots of field-grown maize.
Methods and Results:  Identification and count of bacterial and fungal culturable populations associated to the roots of maize seedlings, changes in culturable community structure according to the richness and diversity indexes concept and shifts in microbial activity through analysis of cellulolytic, ammonification and nitrification potentials were determined, in relation to kernel treatment with biological control agents. Following the treatment of maize kernels with B. amyloliquefaciens at 10⁷ CFU ml⁻¹, an increase in bacterial diversity was observed at the rhizoplane of resultant seedlings. Bacterial richness was significantly increased at the root inner tissues of seedlings treated with Mic. oleovorans . Fusarium , Aspergillus , Penicillium and Trichoderma were the main fungal genera isolated and there population sizes were unequally affected by the addition of biocontrol agents.
Conclusions:  Numbers and types of isolated bacteria and fungi changed in response to the addition of biocontrol agents, while microbial activity remained unchanged with respect to control.
Significance and Impact of the Study:  This study provides an insight of the effects of proven biocontrol agents on micro-organisms naturally associated to the target crop.     

Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.     

Dominance of a clonal green sulfur bacterial population in a stratified lake

For many years, the chemocline of the meromictic Lake Cadagno, Switzerland, was dominated by purple sulfur bacteria. However, following a major community shift in recent years, green sulfur bacteria (GSB) have come to dominate. We investigated this community by performing microbial diversity surveys using FISH cell counting and population multilocus sequence typing [clone library sequence analysis of the small subunit (SSU) rRNA locus and two loci involved in photosynthesis in GSB: fmoA and csmCA ]. All bacterial populations clearly stratified according to water column chemistry. The GSB population peaked in the chemocline ( c . 8 × 10⁶ GSB cells mL⁻¹) and constituted about 50% of all cells in the anoxic zones of the water column. At least 99.5% of these GSB cells had SSU rRNA, fmoA , and csmCA sequences essentially identical to that of the previously isolated and genome-sequenced GSB Chlorobium clathratiforme strain BU-1 (DSM 5477). This ribotype was not detected in Lake Cadagno before the bloom of GSB. These observations suggest that the C. clathratiforme population that has stabilized in Lake Cadagno is clonal. We speculate that such a clonal bloom could be caused by environmental disturbance, mutational adaptation, or invasion.     

Mixed aerobic and anaerobic microbial communities in benzene-contaminated groundwater

Aims:  To investigate the factors affecting benzene biodegradation and microbial community composition in a contaminated aquifer.
Methods and Results:  We identified the microbial community in groundwater samples from a benzene-contaminated aquifer situated below a petrochemical plant. Eleven out of twelve groundwater samples with in situ dissolved oxygen concentrations between 0 and 2·57 mg l⁻¹ showed benzene degradation in aerobic microcosm experiments, whereas no degradation in anaerobic microcosms was observed. The lack of aerobic degradation in the remaining microcosm could be attributed to a pH of 12·1. Three groundwaters, examined by 16S rRNA gene clone libraries, with low in situ oxygen concentrations and high benzene levels, each had a different dominant aerobic (or denitrifying) population, either Pseudomonas , Polaromonas or Acidovorax species. These groundwaters also had syntrophic organisms, and aceticlastic methanogens were detected in two samples. The alkaline groundwater was dominated by organisms closely related to Hydrogenophaga .
Conclusions:  Results show that pH 12·1 is inimical to benzene biodegradation, and that oxygen concentrations below 0·03 mg l⁻¹ can support aerobic benzene-degrading communities.
Significance and Impact of the Study:  These findings will help to guide the treatment of contaminated groundwaters, and raise questions about the extent to which aerobes and anaerobes may interact to effect benzene degradation.     

Bacteria beneath the West Antarctic Ice Sheet

Subglacial environments, particularly those that lie beneath polar ice sheets, are beginning to be recognized as an important part of Earth's biosphere. However, except for indirect indications of microbial assemblages in subglacial Lake Vostok, Antarctica, no sub-ice sheet environments have been shown to support microbial ecosystems. Here we report 16S rRNA gene and isolate diversity in sediments collected from beneath the Kamb Ice Stream, West Antarctic Ice Sheet and stored for 15 months at 4°C. This is the first report of microbes in samples from the sediment environment beneath the Antarctic Ice Sheet. The cells were abundant (∼10⁷ cells g⁻¹) but displayed low diversity (only five phylotypes), likely as a result of enrichment during storage. Isolates were cold tolerant and the 16S rRNA gene diversity was a simplified version of that found in subglacial alpine and Arctic sediments and water. Although in situ cell abundance and the extent of wet sediments beneath the Antarctic ice sheet can only be roughly extrapolated on the basis of this sample, it is clear that the subglacial ecosystem contains a significant and previously unrecognized pool of microbial cells and associated organic carbon that could potentially have significant implications for global geochemical processes.     

两种镉积累型小麦根际微生物群落结构及功能多样性

为探究不同积累型小麦品种对根际微生物群落结构及功能多样性的影响,以镉低积累型小麦济麦22和镉高积累型小麦冀5265为研究材料,采用分离培养法和Biolog-Eco微平板法分析根际细菌数量、可培养优势群落结构以及微生物群落功能多样性。结果表明：污染土壤济麦22根际总细菌数量和抗Cd细菌数量均显著高于冀5265,而非污染土壤中两品种间无差异。污染土济麦22根际发现较多产脲酶和高镉抗性菌株(200 mg/L)。污染土济麦22根际优势菌多为Arthrobacter sp.和Bacillus sp.,冀5265根际优势菌主要为Streptomyces sp.;非污染土济麦22与冀5265根际优势菌群相似,均以Bacillus sp.为主。Biolog试验结果表明,两个小麦品种根际微生物群落对碳源的利用能力存在差异,济麦22根际微生物AWCD值、Mc Intosh指数、Shannon-Wiener指数、Simpson指数在污染土和无污染土中均显著高于冀5265。因此,污染土壤中不同积累型小麦品种根际微生物群落结构及功能多样性均存在差异,该研究结果对于揭示高低积累型小麦根际微生物机制提供了重要参考依...     

Archaeal rRNA diversity and methane production in deep boreal peat

Northern peatlands play a major role in the global carbon cycle as sinks for CO₂ and as sources of CH₄. These diverse ecosystems develop through accumulation of partially decomposed plant material as peat. With increasing depth, peat becomes more and more recalcitrant due to its longer exposure to decomposing processes. Compared with surface peat, deeper peat sediments remain microbiologically poorly described. We detected active archaeal communities even in the deep bottom layers (−220/−280 cm) of two Finnish fen-type peatlands by 16S rRNA-based terminal restriction fragment length polymorphism analysis. In the sediments of the northern study site, all detected archaea were methanogens with Rice Cluster II (RC-II) and Methanosaetaceae as major groups. In southern peatland, Crenarchaeota of a rare unidentified cluster were present together with mainly RC-II methanogens. RNA profiles showed a larger archaeal diversity than DNA-based community profiles, suggesting that small but active populations were better visualized with rRNA. In addition, potential methane production measurements indicated methanogenic activity throughout the vertical peat profiles.     

Use of antibiotic resistance mutations to track strains of obligately anaerobic bacteria introduced into the rumen of sheep

Selective plating procedures were used to follow the fate of rifampicin-resistant mutant strains of the obligately anaerobic species Bacteroides multiacidus and Selenomonas ruminantium after their introduction at numbers around 10⁷/ml into the rumen of sheep. Bacteroides multiacidus strain F100 showed an initially rapid rate of loss (49%/h) but subsequently numbers declined more gradually approaching the limits of detection (10³/ml) after 100 h. Viable cell numbers also decreased in vitro upon addition of F100 cells to whole rumen contents, but remained stable upon addition to cell-free rumen fluid, suggesting protozoal predation. F100 cells were able to grow in vitro in whole rumen contents in the presence of an added utilizable substrate such as sorbitol, but addition of sorbitol to the rumen failed to enhance survival in vivo . In the case of S. ruminantium , introduced rif^R strains persisted in the rumen at levels around 10⁶ ml for at least 30 days.     

Nitrospira-dominated biofilm within a thermal artesian spring: a case for nitrification-driven primary production in a geothermal setting

Water chemistry, energetic modeling, and molecular analyses were combined to investigate the microbial ecology of a biofilm growing in a thermal artesian spring within Hot Springs National Park, AR. This unique fresh water spring has a low dissolved chemical load and is isolated from both light and direct terrestrial carbon input - resulting in an oligotrophic ecosystem limited for fixed carbon and electron donors. Evaluation of energy yields of lithotrophic reactions putatively linked to autotrophy identified the aerobic oxidation of methane, hydrogen, sulfide, ammonia, and nitrite as the most exergonic. Small subunit (SSU) rRNA gene libraries from biofilm revealed a low-diversity microbial assemblage populated by bacteria and archaea at a gene copy ratio of 45:1. Members of the bacterial family 'Nitrospiraceae', known for their autotrophic nitrite oxidation, dominated the bacterial SSU rRNA gene library (approximately 45%). Members of the Thaumarchaeota ThAOA/HWCGIII (>96%) and Thaumarchaeota Group I.1b (2.5%), which both contain confirmed autotrophic ammonia oxidizers, dominated the archaeal SSU rRNA library. Archaea appear to dominate among the ammonia oxidizers, as only ammonia monooxygenase subunit A (amoA) genes belonging to members of the Thaumarchaeota were detected. The geochemical, phylogenetic, and genetic data support a model that describes a novel thermophilic biofilm built largely by an autotrophic nitrifying microbial assemblage. This is also the first observation of 'Nitrospiraceae' as the dominant organisms within a geothermal environment.     

Rapid and reliable DNA extraction and PCR fingerprinting methods to discriminate multiple biotypes of the nematophagous fungus Pochonia chlamydosporia isolated from plant rhizospheres

Aims:  To develop a simple, rapid, reliable protocol producing consistent polymerase chain reaction (PCR) fingerprints of Pochonia chlamydosporia var. chlamydosporia biotypes for analysing different fungal isolates during co-infection of plants and nematodes.
Methods and Results:  DNA extracted from different P. chlamydosporia biotypes was fingerprinted using enterobacterial repetitive intragenic consensus (ERIC)-PCR. Four extraction methods (rapid alkaline lysis; microLYSIS^®-PLUS; DNeasy^®; FTA^® cards) gave consistent results within each protocol but these varied between protocols. Reproducible fingerprints were obtained only if DNA was extracted from fresh fungal cultures that were free of agar. Some DNA degradation occurred during storage, except with the FTA^® cards, used with this fungus for the first time, which provide a method for long-term archiving. Rapid alkaline lysis and ERIC-PCR identified fungal isolates from root and nematode egg surfaces when plants were treated with different combinations of fungal biotypes; the dominant biotype isolated from the rhizosphere was not always the most abundant in eggs.
Conclusions:  ERIC-PCR fingerprinting can reliably detect and identify different P. chlamydosporia biotypes. It is important to use fresh mycelium and the same DNA isolation method throughout each study.
Significance and Impact of the Study:  This evaluation of methods to assess genetic diversity and identify specific P. chlamydosporia biotypes is relevant to other mycelial fungi.     

Seasonal patterns of viruses, bacteria and dissolved organic carbon in a riverine wetland

SUMMARY 1. Viral and bacterial abundances were studied in relation to environmental attributes over an annual period, for both planktonic and attached (sediment, aquatic macrophyte and submerged wood) habitats, in a riverine wetland.
2. Annual mean abundance of planktonic viruses ranged from 2.3 × 10⁵−3.8 × 10⁵ particles mL⁻¹ and varied according to sampling site. Significant seasonal patterns in viral abundance were evident and appeared to be linked to variations in bacterial abundance, dissolved organic carbon and inorganic nutrients.
3. Annual mean abundance of viruses associated with surfaces ranged from 1.3 × 10⁶ particles cm⁻² on aquatic macrophytes to 1.1 × 10⁷ particles cm⁻² on wood and also showed seasonal patterns. The difference in viral dynamics among the different sites emphasizes the importance of considering habitat diversity within wetlands when examining microbial communities.     

Assessing the diversity of AM fungi in arid gypsophilous plant communities

In the present study, we used PCR-Single-Stranded Conformation Polymorphism (SSCP) techniques to analyse arbuscular mycorrhizal fungi (AMF) communities in four sites within a 10 km² gypsum area in Southern Spain. Four common plant species from these ecosystems were selected. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, SSCP analysis, sequencing and phylogenetic analyses. A total of 1443 SSU rRNA sequences were analysed, for 21 AM fungal types: 19 belonged to the genus Glomus , 1 to the genus Diversispora and 1 to the Scutellospora. Four sequence groups were identified, which showed high similarity to sequences of known glomalean species or isolates: Glo G18 to Glomus constrictum , Glo G1 to Glomus intraradices , Glo G16 to Glomus clarum , Scut to Scutellospora dipurpurescens and Div to one new genus in the family Diversisporaceae identified recently as Otospora bareai . There were three sequence groups that received strong support in the phylogenetic analysis, and did not seem to be related to any sequences of AM fungi in culture or previously found in the database; thus, they could be novel taxa within the genus Glomus : Glo G4, Glo G2 and Glo G14. We have detected the presence of both generalist and potential specialist AMF in gypsum ecosystems. The AMF communities were different in the plant studied suggesting some degree of preference in the interactions between these symbionts.     

Microbial community structure in autotrophic nitrifying granules characterized by experimental and simulation analyses

This study evaluates the community structure in nitrifying granules (average diameter of 1600 μm) produced in an aerobic reactor fed with ammonia as the sole energy source by a multivalent approach combining molecular techniques, microelectrode measurements and mathematical modelling. Fluorescence in situ hybridization revealed that ammonia-oxidizing bacteria dominated within the first 200 μm below the granule surface, nitrite-oxidizing bacteria a deeper layer between 200 and 300 μm, while heterotrophic bacteria were present in the core of the nitrifying granule. Presence of these groups also became evident from a 16S rRNA clone library. Microprofiles of NH₄⁺, NO₂^–, NO₃^– and O₂ concentrations measured with microelectrodes showed good agreement with the spatial organization of nitrifying bacteria. One- and two-dimensional numerical biofilm models were constructed to explain the observed granule development as a result of the multiple bacteria–substrate interactions. The interaction between nitrifying and heterotrophic bacteria was evaluated by assuming three types of heterotrophic bacterial growth on soluble microbial products from nitrifying bacteria. The models described well the bacterial distribution obtained by fluorescence in situ hybridization analysis, as well as the measured oxygen, nitrite, nitrate and ammonium concentration profiles. Results of this study are important because they show that a combination of simulation and experimental techniques can better explain the interaction between nitrifying bacteria and heterotrophic bacteria in the granules than individual approaches alone.