The diatom Thalassiosira rotula induces distinct growth responses and colonization patterns of Roseobacteraceae,Flavobacteria and Gammaproteobacteria

Diatoms as important phytoplankton components interact with and are colonized by heterotrophic bacteria. This colonization has been studied extensively in the past but a distinction between the bacterial colonization directly on diatom cells or on the aggregated organic material, exopolymeric substances (EPS), was little addressed. Here we show that the diatom Thalassiosira rotula and EPS were differently colonized by strains of Roseobacteraceae and Flavobacteriaceae in two and tree partner treatments and an enriched natural bacterial community as inoculum. In two partner treatments, the algae and EPS were generally less colonized than in the three partner treatments. Two strains benefitted greatly from the presence of another partner as the proportions of their subpopulations colonizing the diatom cell and the EPS were much enhanced relative to their two partner treatments. Highest proportions of bacteria colonizing the diatom and EPS occurred in the treatment inoculated with the enriched natural bacterial community. Dissolved organic carbon, amino acids and carbohydrates produced by T. rotula were differently used by the bacteria in the two and three partner treatments and most efficiently by the enriched natural bacterial community. Our approach is a valid model system to study physico-chemical bacteria-diatom interactions with increasing complexity.     

Mapping glycoconjugate-mediated interactions of marine Bacteroidetes with diatoms

The degradation of diatoms is mainly catalyzed by Bacteroidetes and this process is of global relevance for the carbon cycle. In this study, a combination of catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) and fluorescent lectin binding analysis (FLBA) was used to identify and map glycoconjugates involved in the specific interactions of Bacteroidetes and diatoms, as well as detritus, at the coastal marine site Helgoland Roads (German Bight, North Sea). The study probed both the presence of lectin-specific extracellular polymeric substances (EPS) of Bacteroidetes for cell attachment and that of glycoconjugates on diatoms with respect to binding sites for Bacteroidetes. Members of the clades Polaribacter and Ulvibacter were shown to form microcolonies within aggregates for which FLBA indicated the presence of galactose containing slime. Polaribacter spp. was shown to bind specifically to the setae of the abundant diatom Chaetoceros spp., and the setae were stained with fucose-specific lectins. In contrast, Ulvibacter spp. attached to diatoms of the genus Asterionella which bound, among others, the mannose-specific lectin PSA. The newly developed CARD-FISH/FLBA protocol was limited to the glycoconjugates that persisted after the initial CARD-FISH procedure. The differential attachment of bacteroidetal clades to diatoms and their discrete staining by FLBA provided evidence for the essential role that formation and recognition of glycoconjugates play in the interaction of bacteria with phytoplankton.     

SUBSTRATUM CONDITIONING AND DIATOM COLONIZATION IN DIFFERENT CURRENT REGIMES1

Short-term (3 day) diatom colonization of clean ceramic tiles and tiles conditioned with a thin, nonalgal biofilm was examined in fast- and slow-current outdoor experimental stream channels to assess effects of organic conditioning and current regime on diatom colonization. Colonization rates onto unconditioned tiles were 10 times lower in fast current that in slow. Substratum conditioning reduced the effects of current because organic conditioning of tiles significantly enhanced colonization in fast current, but not in slow current. Two diatom taxa (Nitzschia acicularis W. Sm. and Synedra radians Kütz) colonized unconditioned tiles more rapidly than conditioned tiles in slow current, suggesting the existence of negative interactions between these diatoms and bacteria and/or organics in conditioning films.     

Influence of bacteria and salinity on diatom biogenic silica dissolution in estuarine systems

Dissolution of diatom biogenic silica (bSiO₂) in estuaries and its control by water salinity and bacteria were investigated using the river euryhaline species Cyclotella meneghiniana as a model. Laboratory-controlled bioassays conducted at different salinities with an estuarine bacteria inoculum showed a faster dissolution of diatom bSiO₂ at the lowest salinity where bacteria were the most abundant. However in another experiment, salinity increase clearly enhanced the dissolution of cleaned frustules (organic matter free). The presence of active bacteria might therefore predominate on the effect of salinity for freshly lysed diatoms whereas salinity might rather control dissolution of organic-matter-free frustule remains. Incubation of cultivated diatoms at different protease concentrations revealed that high proteolytic activities had little effect on bSiO₂ dissolution at a 1-month scale in spite of an efficient removal of organic matter from the frustules. Altogether it is hypothesized that bacterial colonization increases bSiO₂ dissolution by creating a microenvironment at the diatom surface with high ectoproteolytic activity but also via the release of metabolic byproducts since the presence of organic matter seems generally to facilitate diatom bSiO₂ dissolution.     

Biofilm and capsule formation of the diatom Achnanthidium minutissimum are affected by a bacterium

Photoautotrophic biofilms play an important role in various aquatic habitats and are composed of prokaryotic and/or eukaryotic organisms embedded in extracellular polymeric substances (EPS). We have isolated diatoms as well as bacteria from freshwater biofilms to study organismal interactions between representative isolates. We found that bacteria have a strong impact on the biofilm formation of the pennate diatom Achnanthidium minutissimum. This alga produces extracellular capsules of insoluble EPS, mostly carbohydrates (CHO), only in the presence of bacteria (xenic culture). The EPS themselves also have a strong impact on the aggregation and attachment of the algae. In the absence of bacteria (axenic culture), A. minutissimum did not form capsules and the cells grew completely suspended. Fractionation and quantification of CHO revealed that the diatom in axenic culture produces large amounts of soluble CHO, whereas in the xenic culture mainly insoluble CHO were detected. For investigation of biofilm formation by A. minutissimum, a bioassay was established using a diatom satellite Bacteroidetes bacterium that had been shown to induce capsule formation of A. minutissimum. Interestingly, capsule and biofilm induction can be achieved by addition of bacterial spent medium, indicating that soluble hydrophobic molecules produced by the bacterium may mediate the diatom/bacteria interaction. With the designed bioassay, a reliable tool is now available to study the chemical interactions between diatoms and bacteria with consequences for biofilm formation.     

Dynamics of diatom colonization process in some rivers influenced by urban pollution (Hanoi, Vietnam)

Periphytic diatom communities’ colonization patterns were studied at three sampling stations of the Red–Nhue–Tolich hydrosystem presenting different urban pollution levels by using artificial substrates for 6 weeks in dry season 2005. Structural characteristics of periphytic diatoms developed on glass substrates at each sampling site were followed and compared. This experiment showed, through various general criteria (total diatom density, dry weight biomass) and specific criteria (relative diatom abundances, indices) that the structure of benthic diatoms developed on these substrates was strongly affected by pollution as early as the second week of colonization. Communities exposed to heavily and moderately polluted sites contained species which are known to be saprophilous or tolerant to organic pollution such as Nitzschia umbonata, Nitzschia palea, Cyclotella meneghiniana, Eolimna minima. Growth inhibition of diatom communities at the heavily polluted site was mostly related to a strong increase of organic load rather than to low metallic input, though metallic (Cd and Zn) burdens have been successfully quantified in the biofilms developed at the three studied sites. Nevertheless, no significant difference in species richness and diversity index between colonization duration times was observed. Based on values of diatom indices Indice de Polluosensibilité (IPS) and diatom assemblage index to organic water pollution (DAIPo), water quality could be classified as medium quality at Red site, polluted at NT₂ and heavily polluted at TL. Thus, the use of diatoms as a tool for water assessment appears suitable for monitoring rivers in Vietnam, as it is in several other countries.     

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump.     

The biofilm matrix of Campylobacter jejuni determined by fluorescence lectin-binding analysis

Campylobacter jejuni is responsible for the most common bacterial foodborne gastroenteritis. Despite its fastidious growth, it can survive harsh conditions through biofilm formation. In this work, fluorescence lectin-binding analysis was used to determine the glycoconjugates present in the biofilm matrix of two well-described strains. Screening of 72 lectins revealed strain-specific patterns with six lectins interacting with the biofilm matrix of both strains. The most common sugar moiety contained galactose and N-acetylgalactosamine. Several lectins interacted with N-acetylglucosamine and sialic acid, probably originated from the capsular polysaccharides, lipooligosaccharides and N-glycans of C. jejuni. In addition, glycoconjugates containing mannose and fucose were detected within the biofilm, which have not previously been found in the C. jejuni envelope. Detection of thioflavin T and curcumin highlighted the presence of amyloids in the cell envelope without association with specific cell appendages. The lectins ECA, GS-I, HMA and LEA constitute a reliable cocktail to detect the biofilm matrix of C. jejuni.     

GROWTH OF VITAMIN B12-REQUIRING MARINE DIATOMS IN MIXED LABORATORY CULTURES WITH VITAMIN B12-PRODUCING MARINE BACTERIA12

The vitamin B₁₂ requirement of several marine diatoms can be satisfied in B₁₂^?limited laboratory cultures by heterotrophic marine bacteria isolated from the same waters and from sediments. The bacteria can utilize diatom excretory products, or the remains of dead diatom cells, in the production of the vitamin. The growth of 12 B₁₂¹? requiring diatoms (7 genera) in mixed cultures with 14 different bacteria (without added B₁₂) was compared to the growth of those same diatoms in axenic cultures with excess added B₁₂. Diatom growth was generally rapid in the first few days, followed by sustained, slower growth. The diatom yields in mixed cultures ranged from 0.8 to 84% of the yields in axenic cultures with added B₁₂. In a detailed study of one mixed culture, increases in diatom densities were paralleled by increases in cell densities of the bacterium during the first few days of exponential diatom growth. During the period of slow diatom growth, when diatom densities oscillated but steadily increased, the decreases in diatom densities were associated with increased bacterial growth. This suggests that death of a fraction of the B₁₂-limited diatom population releases sufficient organic matter to stimulate growth of the bacteria and their subsequent excretion of B₁₂; this B₁₂ in turn stimulates further growth of the diatoms. Diatom-bacteria interactions leading to the production of B₁₂ may be important in maintaining viable populations of B₁₂-requiring diatoms in nutrient-poor waters during periods between blooms, when conditions are unfavorable for rapid growth.     

Diversity and distribution of epibiotic bacteria on Pseudo-nitzschia multiseries (Bacillariophyceae) in culture, and comparison with those on diatoms in native seawater

Previous studies on the interaction between bacteria and harmful algal bloom species have mostly considered the bacteria in the bulk solution. Here, we document the abundance and mode of attachment of bacteria growing on the cell surface of the domoic acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle in culture, compared with diatoms in field samples. The epiphytic bacteria were examined by scanning electron microscopy to visualize their morphology and mode of attachment. Two P. multiseries cultures were studied: clone CLN-1 and sub-clone CLN-1-NRC; the latter had been maintained in another laboratory for 2 years. Each of these P. multiseries cultures exhibited a clearly different assemblage of epibiotic bacteria, even though both originated from the same parent culture. The bacterial diversity was greater in clone CLN-1 (nine distinct morphotypes seen) than in sub-clone CLN-1-NRC (six morphotypes). The former clone also produced more domoic acid than the latter. There was a succession of bacterial morphotypes as well as an increase in the number of epiphytic bacteria per diatom cell during the progression from exponential to stationary phase. The most diverse and common morphotypes were rod-shaped cells (e.g. a Caulobacter-like bacterium attached by a discoid holdfast). Epibionts showed a preference for attachment at specific regions of the host diatom frustule, e.g. the raphe or cingulum, locations where organic matter may be extruding from the diatom cell. Most diatom cells carried only one to five bacteria, and up to ca. 60% of the intact diatom cells (although intact cells themselves were infrequent) were still free of epibiotic bacteria at the end of the 31-day batch culture experiment. Sequencing of the SSU rRNA gene showed that five of the eight bacterial strains isolated from the P. multiseries cultures were members of the Alphaproteobacteria, three of the Gammaproteobacteria and one of the Bacteroidetes. A morphologically diverse assemblage of epibiotic bacteria was also found on both centric and pennate planktonic diatoms in natural coastal waters. Of the eight morphotypes recorded, all but two were also found in the cultures. Relatively fewer wild diatom cells carried bacteria compared to cells in culture. We hypothesize that the diversity and abundance of epiphytic bacteria may explain some of the variability seen in the production of DA by different P. multiseries clones, and should be considered as another important and controllable variable that influences diatom cell physiology.     

Lectins and also bacteria modify the glycosylation of gut surface receptors in the rat

Oral exposure to lectins or the presence or absence of bacteria in the rat small intestine were shown by histological methods using anti-lectin antibodies or digoxigenin-labelled lectins to have major effects on the state of glycosylation of lumenal membranes and cytoplasmic glycoconjugates of epithelial cells. Taken together with the dramatic effects of exposure to lectins on gut function, metabolism and bacterial ecology, this can be used as a basis for new perspectives of biomedical manipulations to improve health. Abbreviations: DIG, digoxigenin-labelled; POD, peroxidase-labelled; Spf, specific pathogen-free; TBS, Saline (0.9% w/v NaCl solution) buffered with 0.05m Tris-glycine, pH 7.8; PAP, antibody-peroxidase-antiperoxidase. For lectins see Table 1.     

Antidiatom and antibacterial activity of epiphytic bacteria isolated from <Emphasis Type="Italic">Ulva</Emphasis><Emphasis Type="Italic">lactuca</Emphasis> in tropical waters

Bacteria and diatoms are primary colonizers of marine surfaces and hence play a crucial role in the attachment and subsequent growth of macroorganisms. It has been suggested that the temperate green alga Ulva lactuca relies on the defence provided by the epiphytic bacterial community to regulate surface fouling of colonising organisms. In this study, ten resident bacterial isolates from tropical U. lactuca were tested for their antibacterial and antidiatom properties that may regulate surface colonization on the algae. Sixty percent of the epiphytic isolates expressed antibacterial properties against other resident bacteria and 80% had antidiatom activity against the pennate diatom, Cylindrotheca fusiformis. Isolates of the Pseudoalteromonas genus showed both- antibacterial and antidiatom activities, while members of the genus Bacillus, Vibrio and Shewanella mostly possessed antidiatom activity. Our results show that a high proportion of bacterial isolates from tropical U. lactuca, like that of their temperate counterparts contain antibiotic properties that might impact on the bacterial community composition and prevent fouling by diatoms.     

Bacteria may induce the secretion of mucin‐like proteins by the diatom Phaeodactylum tricornutum

Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as a sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin‐like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline‐, serine‐, threonine‐rich (PST) domains in these proteins were also found in combination with protease‐, glucosidase‐ and leucine‐rich repeat‐domains. Bioinformatic functional predictions indicate that several of these newly identified diatom‐specific proteins may be involved in algal defense, intercellular signaling, and aggregation.     

SEASONALITY OF MICROBIAL FOULING ON ASCOPHYLLUM NODOSUM (L.) LEJOL., FUCUS VESICULOSUS L., POLYSIPHONIA LANOSA (L.) TANDY AND CHONDRUS CRISPUS STACKH1

The nature and seasonal extent of microbial fouling on Ascophyllum nodosum (L.) LeJol., Fucus vesiculosus L., Polysiphonia lanosa (L.) Tandy and Chondrus crispus Stackh. were observed by scanning electron microscopy. Bacterial filaments and smaller rod and coccoid forms dominated the fouling communities on all species, with pennate diatoms constituting a minor component in contrast to results with plastic substrates on which pennate diatoms dominated and preceded bacterial colonization. The total percent microbial coverage on the surfaces of all four seaweed species was determined by monthly stereological analyses of representative composite micrographs. These showed a simultaneous decline between April and May which could represent the die-off of the cold water bacterial flora when water temperature increased past the threshold for obligate psychrophiles. Microbial colonization patterns were directly correlated (P = 0.005) with maximum coverage in April and November–December and reduced levels from May to October. Significant inverse (P < 0.041) correlations between total percent coverage and water temperature indicate distinct seasonal cycles, however, the patterns of dominance by filamentous bacteria and rod and coccoid forms were markedly different. Total coverage patterns of both rhodophytes showed no apparent seasonal cycle and were not related to water temperature. Rod and coccoid bacteria were apparently suppressed year round on P. lanosa relative to the other species. These interspecific differences in seasonal fouling patterns are discussed in light of possible modes of regulation, especially algal antibiosis.     

Microbial Response of a Freshwater Benthic Community to a Simulated Diatom Sedimentation Event: Interactive Effects of Benthic Fauna

Abstract The response of a sediment microbial assemblage to a pulse of diatoms was studied over 36 days by measuring bacterial activity and biomass, ATP concentration, and overall community respiration in laboratory microcosms. Also, the contribution of macrofaunal chironomids to the decomposition of settling diatoms in benthic communities, and the relative importance of benthic meiofauna in community metabolism, were determined. The addition of diatoms resulted in an immediate response by sediment bacteria, with higher bacterial production recorded after only 2 h, and a more than tenfold increase within one day. The rapid response by sediment bacteria was accompanied by relatively high initial concentrations of dissolved organic carbon. In treatments receiving diatoms, higher bacterial production was sustained throughout the experiment. Surprisingly, neither these elevated production estimates, nor the starvation of controls affected bacterial abundance. Mean bacterial cell volume, however, was markedly affected by the addition of diatoms. Combining community respiration measurements and bacterial production estimates showed that growth efficiencies for sediment bacteria ranged from 14.6 to 34.5%. The contribution of ambient meiozoobenthos to carbon metabolism was less than 1%. Carbon budgets showed that 1.3 mg C was cooxidized along with 4.3 mg added diatom C. Sediment reworking by Chironomus larvae initially enhanced bacterial production, but the presence of Chironomus resulted in lower bacterial production estimates after 16 and 36 days. This was interpreted as a result of faster decomposition of diatoms in treatments with chironomids, which was validated by a faster decline of ATP and chlorophyll a in the sediment. Our results indicate that Chironomus larvae compete with sediment bacteria for available organic substrates. Received: 11 June 1996; Accepted: 13 August 1996     

Host tissues as microhabitats for Wolbachia and quantitative insights into the bacterial community in terrestrial isopods

Animal–bacterial symbioses are highly dynamic in terms of multipartite interactions, both between the host and its symbionts as well as between the different bacteria constituting the symbiotic community. These interactions will be reflected by the titres of the individual bacterial taxa, for example via host regulation of bacterial loads or competition for resources between symbionts. Moreover, different host tissues represent heterogeneous microhabitats for bacteria, meaning that host‐associated bacteria might establish tissue‐specific bacterial communities. Wolbachia are widespread endosymbiotic bacteria, infecting a large number of arthropods and filarial nematodes. However, relatively little is known regarding direct interactions between Wolbachia and other bacteria. This study represents the first quantitative investigation of tissue‐specific Wolbachia–microbiota interactions in the terrestrial isopod Armadillidium vulgare. To this end, we obtained a more complete picture of the Wolbachia distribution patterns across all major host tissues, integrating all three feminizing Wolbachia strains (wVulM, wVulC, wVulP) identified to date in this host. Interestingly, the different Wolbachia strains exhibited strain‐specific tissue distribution patterns, with wVulM reaching lower titres in most tissues. These patterns were consistent across different host genetic backgrounds and might reflect different co‐evolutionary histories between the Wolbachia strains and A. vulgare. Moreover, Wolbachia‐infected females carried higher total bacterial loads in several, but not all, tissues, irrespective of the Wolbachia strain. Taken together, this quantitative approach indicates that Wolbachia is part of a potentially more diverse bacterial community, as exemplified by the presence of highly abundant bacterial taxa in the midgut caeca of several A. vulgare populations.     

Quorum sensing regulates ‘swim-or-stick’ lifestyle in the phycosphere

Interactions between phytoplankton and bacteria play major roles in global biogeochemical cycles and oceanic nutrient fluxes. These interactions occur in the microenvironment surrounding phytoplankton cells, known as the phycosphere. Bacteria in the phycosphere use either chemotaxis or attachment to benefit from algal excretions. Both processes are regulated by quorum sensing (QS), a cell–cell signalling mechanism that uses small infochemicals to coordinate bacterial gene expression. However, the role of QS in regulating bacterial attachment in the phycosphere is not clear. Here, we isolated a Sulfitobacter pseudonitzschiae F5 and a Phaeobacter sp. F10 belonging to the marine Roseobacter group and an Alteromonas macleodii F12 belonging to Alteromonadaceae, from the microbial community of the ubiquitous diatom Asterionellopsis glacialis. We show that only the Roseobacter group isolates (diatom symbionts) can attach to diatom transparent exopolymeric particles. Despite all three bacteria possessing genes involved in motility, chemotaxis, and attachment, only S. pseudonitzschiae F5 and Phaeobacter sp. F10 possessed complete QS systems and could synthesize QS signals. Using UHPLC–MS/MS, we identified three QS molecules produced by both bacteria of which only 3-oxo-C_16:1-HSL strongly inhibited bacterial motility and stimulated attachment in the phycosphere. These findings suggest that QS signals enable colonization of the phycosphere by algal symbionts.     

CONTINUOUS FLOW CULTURE OF BENTHIC DIATOMS AND ITS APPLICATION TO BIOASSAY1,2

The benthic diatom, Navicula seminulum var. hustedtii Patr, was isolated from the field and studied in laboratory cultures. Experiments were conducted to determine the suitability of Millipore membranes and solidified agar substrate for diatom colonization under continuous flow conditions. Diatom colonization showed large variation and low reproducibility on Millipore membranes of different materials and pore sizes. Solidified agar substrate supported stable and reproducible colonization and is nutritionally neutral, translucent, homogeneous and easy to sample. The diatom colonization process on agar substrate involved four growth phases: i) pioneer; ii) exponential; iii) steady state; and, iv) vanishing. The culture system was also used in bioassay, testing the toxic effect of copper on the growth of benthic diatoms. The proposed method provides a useful means for studying autecology of benthic diatoms as well as for bioassay work.     

Diatoms in Acid Mine Drainage and Their Role in the Formation of Iron-Rich Stromatolites

Adverse conditions in the acid mine drainage (AMD) system at the Green Valley mine, Indiana, limit diatom diversity to one species, Nitzschia tubicola. It is present in three distinct microbial consortia: Euglena mutabilis-dominated biofilm, diatomdominated biofilm, and diatom-exclusive biofilm. E. mutabilis dominates the most extensive biofilm, with lesser numbers of N. tubicola, other eukaryotes, and bacteria. Diatom-dominated biofilm occurs as isolated patches containing N. tubicola with minor fungal hyphae, filamentous algae, E. mutabilis, and bacteria. Diatom-exclusive biofilm is rare, composed entirely of N. tubicola.

Diatom distribution is influenced by seasonal and intraseasonal changes in water temperature and chemistry. Diatoms are absent in winter due to cool water temperatures. In summer, isolated patchy communities are present due to warmer water temperatures. In 2001, the diatom community expanded its distribution following a major rainfall that temporarily diluted the effluent, creating hospitable conditions for diatom growth. After several weeks when effluent returned to preexisting conditions, the diatom biofilm retreated to isolated patches, and E. mutabilis biofilm flourished.

Iron-rich stromatolites underlie the biofilms and consist of distinct laminae, recording spatial and temporal oscillations in physicochemical conditions and microbial activity. The stromatolites are composed of thin, wavy laminae with partially decayed E. mutabilis biofilm, representing microbial activity and iron precipitation under normal AMD conditions. Alternating with the wavy layers are thicker, porous, spongelike laminae composed of iron precipitated on and incorporated into radiating colonies of diatoms. These layers indicate episodic changes in water chemistry, allowing diatoms to temporarily dominate the system.