Biofilm Response Research Articles

Extracellular polymeric substances affect the responses of multi-species biofilms in the presence of sulfamethizole.

The occurrence and transportation of antibiotics in biofilms from natural and engineered sources have attracted increasing interests. Nevertheless, the effects of extracellular polymeric substances (EPS) on the responses of biofilms to the exposure to antibiotics are not clear. In this study, the effects of EPS on the sorption and biological responses to one representative antibiotic, sulfamethizole (STZ), in model biofilms were investigated. Proteins dominated the interactions between the EPS and the STZ and the EPS from a moving bed biofilm reactor exhibited the strongest interaction with the STZ. The EPS served as important reservoirs for the STZ and the tested biofilms all showed reduced sorption capacities for the STZ after the EPS were extracted. The respiratory rates and typical enzymatic activities were reduced after the EPS were extracted. High-throughput 16S rRNA gene sequencing results confirmed that the bacterial community in the biofilm without the EPS was more vulnerable to antibiotic shock as indicated by the community diversity and richness indices. A greater increase in the abundance of susceptible species was observed in the natural biofilm. The results comprehensively suggested that the EPS played important role in biosorption of STZ and alleviated the direct damage of the antibiotic to the cells; in addition the extent of the bacterial community response was associated with the origins of the biofilms. Our study provided details on the responses of multi-species biofilms to the exposure to an antibiotic and highlighted the role of the EPS in interacting with the antibiotic, thereby providing a deeper understanding of the bioremediation of antibiotics in real-life natural and engineered biofilm systems.

Investigation of Differentially Expressed Proteins of Candida tropicalis Biofilm in Response to Citral

Candida tropicalis is an opportunistic human pathogen with an ability to cause superficial as well as systemic infections in immunocompromised patients. C. tropicalis biofilms can cause persistent infections which are difficult to treat due to acquired resistance. Citral has been used as antifungal agents against Candida species and biofilms. In the present study, we used one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF-MS) to identify the changes in the protein expression of C. tropicalis in response to the sub-lethal concentration of citral. A total of six differential proteins involved inoxidative stress (Tsa1p, Psa2p), amino acid biosynthesis (Met6p, Gln1p), heme biosynthesis (Hem13p) and glucose metabolism (Eno1p) pathways were detected. Our results revealed citral-induced proteins of C. tropicalis biofilm. This study will further help in the interpretation of mode of action of citral and development of novel antifungal agents against these potential protein targets.

The stringent response promotes biofilm dispersal in Pseudomonas putida

Biofilm dispersal is a genetically programmed response enabling bacterial cells to exit the biofilm in response to particular physiological or environmental conditions. In Pseudomonas putida biofilms, nutrient starvation triggers c-di-GMP hydrolysis by phosphodiesterase BifA, releasing inhibition of protease LapG by the c-di-GMP effector protein LapD, and resulting in proteolysis of the adhesin LapA and the subsequent release of biofilm cells. Here we demonstrate that the stringent response, a ubiquitous bacterial stress response, is accountable for relaying the nutrient stress signal to the biofilm dispersal machinery. Mutants lacking elements of the stringent response – (p)ppGpp sythetases [RelA and SpoT] and/or DksA – were defective in biofilm dispersal. Ectopic (p)ppGpp synthesis restored biofilm dispersal in a ∆relA ∆spoT mutant. In vivo gene expression analysis showed that (p)ppGpp positively regulates transcription of bifA, and negatively regulates transcription of lapA and the lapBC, and lapE operons, encoding a LapA-specific secretion system. Further in vivo and in vitro characterization revealed that the PbifA promoter is dependent on the flagellar σ factor FliA, and positively regulated by ppGpp and DksA. Our results indicate that the stringent response stimulates biofilm dispersal under nutrient limitation by coordinately promoting LapA proteolysis and preventing de novo LapA synthesis and secretion.

Response of anodic biofilm to hydrodynamic shear in two-chamber bioelectrochemical systems

Hydrodynamics plays an important role in the design and operation of bioreactors for wastewater treatment. In this work, the response of anodic biofilm to hydrodynamic shear in a common two-chamber bioelectrochemical system (BES) was investigated. The instantaneous flow field in the anodic compartment of the BES was obtained through particle image velocimetry. The effect of anodic hydrodynamic shear on the BES performance in terms of current density, coulombic efficiency and internal resistance was evaluated successively. In addition, the microbial communities of anodic biofilm that alter with hydrodynamic changes were revealed. The results show that the flow regime involving with vortex and dead zone in the anode compartment of the BES was complicated. An optimum hydrodynamic shear existed to yield the maximum stable current density and coulombic efficiency as well as the lowest anodic internal resistance for the BES. Moreover, the microbial communities of the anodic biofilm were sensitive to the hydrodynamic shear. The largest biomass of the biofilm attached on the electrode surface and the highest relative abundance of electrochemically active bacterium, such as dominant Desulfovibrio and Geobacter, were observed under the optimum hydrodynamic conditions.

The integrative effect of periphyton biofilm and tape grass (Vallisneria natans) on internal loading of shallow eutrophic lakes.

The response of periphyton biofilm and the submerged macrophyte tape grass (Vallisneria natans) to internal loading from eutrophic lake sediments were evaluated in microcosms. The sediments from the littoral zone and center of a lake were selected to carry out the microcosm experiment. To determine how the differences in the periphyton biofilm and V. natans growth alone or in combination, we measured changes in water quality, growth, and TP in the periphyton biofilm and V. natans in microcosms containing these sediments. The results showed that the average daily TN and TP removal rates were 32.6 and 35.4%, respectively, in the microcosms containing the lake center sediments by V. natans and the periphyton biofilm. The presence of the periphyton biofilm and V. natans increased the pH, dissolved oxygen, and redox potential and decreased the conductivity in the overlying water in all treatments. Compared to the state before the treatments, V. natans grew well, with a significant increase in biomass (3.1- to 5.5-fold growth) and TP amount (5.1- to 8.8-fold) in all treatments after 48days. However, the growth of V. natans that combined with the periphyton biofilm was better than that of V. natans alone, as reflected by the dry weight, chlorophyll a content, malondialdehyde content, and TP amount. In conclusion, the periphyton biofilm was beneficial for the growth of V. natans, and the appropriate combination of V. natans and periphyton biofilm would be a potential method for the ecological restoration of eutrophic lakes.

Function of biofilms in phytopathogenic bacterial-host interactions

Biofilms are complex three-dimensional bacterial assemblages that attach to biotic or abiotic solid surfaces, and frequently embed within a self-produced matrix of extracellular polymeric substances. Biofilm formation is a microbial defense response to biotic and abiotic stresses, and a key factor for survival in adverse environments. A wide variety of microorganisms can colonize distant tissues of higher plants, such as leaves, vascular network and roots, and adhere to the surface of the tissues to form biofilms. The dynamic processes in forming biofilms in response to plant internal environment are key steps required for full virulence of phytopathogenic bacteria. Exploring the mechanisms involved in regulation of bacterial biofilms is important for understanding the plant-pathogens interactions. In this review, we summarized the research progresses related to the biofilms of bacterial phytopathogens, including biofilm characteristics, essential regulatory mechanisms and key signals affecting the transition between a planktonic lifestyle and multicellular behavior.

Biofilms Benefiting Plants Exposed to ZnO and CuO Nanoparticles Studied with a Root-Mimetic Hollow Fiber Membrane.

Plants exist with a consortium of microbes that influence plant health, including responses to biotic and abiotic stress. While nanoparticle (NP)-plant interactions are increasingly studied, the effect of NPs on the plant microbiome is less researched. Here a root-mimetic hollow fiber membrane (HFM) is presented for generating biofilms of plant-associated microbes nurtured by artificial root exudates (AREs) to correlate exudate composition with biofilm formation and response to NPs. Two microbial isolates from field-grown wheat, a bacillus endophyte and a pseudomonad root surface colonizer, were examined on HFMs fed with AREs varying in N and C composition. Bacterial morphology and biofilm architecture were characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM) and responses to CuO and ZnO NP challenges of 300 mg/L evaluated. The bacillus isolate sparsely colonized the HFM. In contrast, the pseudomonad formed robust biofilms within 3 days. Dependent on nutrient sources, the biofilm cells produced extensive extracellular polymeric substances (EPS) and large intracellular granules. Pseudomonad biofilms were minimally affected by ZnO NPs. CuO NPs, when introduced before biofilm maturation, strongly reduced biofilm formation. The findings demonstrate the utility of the HFM root-mimetic to study rhizoexudate influence on biofilms of root-colonizing microbes but without active plant metabolism. The results will allow better understanding of how microbe-rhizoexudate-NP interactions affect microbial and plant health.

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OBJECTIVES/SPECIFIC AIMS: The specific objectives of this project are (1) identify, test, and validate the parameters for a simplified NLOM imaging probe that will provide specific research and point-of-care information on biofilm presence, therapeutic need and response of individual wounds to treatment. (2) Identify specific proteomic and metabolomic biomarkers of (i) wound susceptibility to infection, (ii) wound response to the most commonly used antibacterial measures in wounds, and (iii) establish criteria for more effective interventions. METHODS/STUDY POPULATION: First, optimal use parameters for NLOM including illumination, field of view, focal length, linear Versus concentric image acquisition, detection and filter wavelengths were identified. Parameters for evaluation included ease and speed of imaging, ability to map diagnostic criteria. Next, using the optimised NLOM imaging modality in bacterial biofilm isolates and subsequently a rabbit ear model of biofilm wound infection, proteomic and metabolomic biomarkers of susceptibility to infection were identified. The effects of 2 standard debridement and anti-infective treatments, polyvidone-iodine solution or cetrimide 15%+ chlorhexidine gluconate 1.5% were mapped in situ for up to 10 days using the NLOM probe. RESULTS/ANTICIPATED RESULTS: Using the novel custom NLOM probe, high resolution mapping of wound biofilm infection, as well as the underlying tissue was performed throughout the onset, development, treatment, and resolution of wound biofilm infection. Specific microbiological, microstructural, oxygenation, and pH parameters were mapped at defined surface and subsurface locations and time-points. Findings included the determination that some standard antimicrobial formulations provide a supportive environment for wound infection, and that micro-channels within the biofilm and their interface with the tissues serve as an important predictor and indicator of wound infection establishment, progression, and response. DISCUSSION/SIGNIFICANCE OF IMPACT: The novel multimodality in vivo NLOM imaging approach establishes an important tool for earlier and more specific diagnosis of wound infection risk, virulence, and invasiveness along with markers of successful treatment, and a simple clinical imaging tool for improving wound infection prevention and treatment.

Dynamics of Mixed–Candida Species Biofilms in Response to Antifungals

Oral infections caused by Candida species, the most commonly isolated human fungal pathogen, are frequently associated with biofilms. Although Candida albicans is the predominant organism found in patients with oral thrush, a biofilm infection, there is an increasing incidence of oral colonization and infections caused by non-albicans Candida species, including C. glabrata, C. dubliniensis, and C. tropicalis, which are frequently more resistant to antifungal treatment. While single-species Candida biofilms have been well studied, considerably less is known about the dynamics of mixed–Candida species biofilms and how these dynamics are altered by antifungal treatment. To address these questions, we developed a quantitative polymerase chain reaction–based approach to determine the precise species composition of mixed–Candida species biofilms formed by clinical isolates and laboratory strains in the presence and absence of clinically relevant concentrations of 3 commonly used antifungals: fluconazole, caspofungin, and amphotericin B. In monospecies biofilms, fluconazole exposure favored growth of C. glabrata and C. tropicalis, while caspofungin generally favored significant growth of all species to a varying degree. Fluconazole was not effective against preformed mixed–Candida species biofilms while amphotericin B was potent. As a general trend, in mixed–Candida species biofilms, C. albicans lost dominance in the presence of antifungals. Interestingly, presence in mixed versus monospecies biofilms reduced susceptibility to amphotericin B for C. tropicalis and C. glabrata. Overall, our data suggest that antifungal treatment favors the growth of specific non-albicans Candida species in mixed–Candida species biofilms.

Acid tolerance properties of dental biofilms in vivo

BackgroundThe ecological plaque hypothesis explains caries development as the result of the enrichment of acid tolerant bacteria in dental biofilms in response to prolonged periods of low pH. Acid production by an acid tolerant microflora causes demineralisation of tooth enamel and thus, individuals with a greater proportion of acid tolerant bacteria would be expected to be more prone to caries development. Biofilm acid tolerance could therefore be a possible biomarker for caries prediction. However, little is known about the stability of biofilm acid tolerance over time in vivo or the distribution throughout the oral cavity. Therefore the aim of this study was to assess intra-individual differences in biofilm acid-tolerance between different tooth surfaces and inter-individual variation as well as stability of acid tolerance over time.ResultsThe majority of the adolescents showed low scores for biofilm acid tolerance. In 14 of 20 individuals no differences were seen between the three tooth sites examined. In the remaining six, acid-tolerance at the premolar site differed from one of the other sites. At 51 of 60 tooth sites, acid-tolerance at baseline was unchanged after 1 month. However, acid tolerance values changed over a 1-year period in 50% of the individuals.ConclusionsBiofilm acid tolerance showed short-term stability and low variation between different sites in the same individual suggesting that the acid tolerance could be a promising biological biomarker candidate for caries prediction. Further evaluation is however needed and prospective clinical trials are called for to evaluate the diagnostic accuracy.

Sustainable pollutant removal by periphytic biofilm via microbial composition shifts induced by uneven distribution of CeO2 nanoparticles

The responses of periphytic biofilm to CeO2 nanoparticle (CNP) exposure were explored by investigating community shifts and pollutant removal. Results showed that CNPs entered the sensitive microbial cells in the periphytic biofilm, leading to cytomembrane damage and intracellular reactive oxygen species (ROS) generation. The periphytic biofilm communities were, however, able to adapt to the prolonged exposure and maintain their pollutant removal (i.e., phosphorus, nitrogen and copper, organic matter) performance. Observations under synchrotron radiation scanning transmission X-ray microscopy revealed that fewer CNPs were distributed in algal cells compared to bacterial cells, wherein the transformation between Ce(IV) and Ce(III) occurred. High-throughput sequencing further showed that the proportion of algae, such as Leptolyngbya and Nostoc, significantly increased in the periphytic biofilm exposed to CNPs while the proportion of bacteria, such as Bacilli and Gemmatimonadetes, decreased. This change in community composition might be the primary reason for the sustained pollutant removal performance of the periphytic biofilm.

The Immune Response in Periodontal Tissues.

The uniqueness of periodontal diseases is caused by several factors. This group of diseases is caused by numerous bacterial species formed in the dental biofilm, and one cannot distinguish the specific pathogen that is responsible for the disease initiation or progress (though Gram-negative anaerobic rods are associated with the advanced form of the disease). The disease is both infectious and inflammatory in its nature, and in the state of health there is always a subclinical level of inflammatory response, caused by the so-called harmless bacteria. Negligence in oral hygiene may result in maturation of the biofilm and trigger host response, manifesting clinically as gingivitis or-later and in susceptible subjects-as periodontitis. The article presents the contemporary knowledge of the inflammatory reaction occurring in tissues surrounding the tooth during periodontal inflammation. The most important mechanisms are described, together with implications for clinicists.

Staphylococcus aureus infections, some second thoughts.

Staphylococcus aureus (S. aureus) is well known for its ability to cause life-threatening infections. On the other hand, this bacterium can thrive as a commensal on and in human tissues without causing much problems. How big a threat is S. aureus actually? Furthermore, commensalism is associated with biofilms, where can we find them, and which natural and artificial components activate biofilm formation? Recent findings on S. aureus carriage on skin, mucosa, and in wounds indicate the presence of large numbers of S. aureus, yet its abundance can be without major implications for the host. S. aureus is often present in biofilms, together with other microorganisms, which can stimulate biofilm formation of S. aureus, in addition medicine including antibiotics can do the same. S. aureus can cause devastating infections, but when we take into consideration the ubiquitous presence of S. aureus, the risk seems to be relatively low. S. aureus forms biofilms in response to the 'hazards' on the human body, and signal to do so can come from various sources. All this has to be taken into consideration when we treat a patient as this might have enormous impact on the outcome.

Biofilms and Oxidizing Biocides; Evaluation of Disinfection and Removal Effects by Using Established Microbial Systems.

The formation of bacterial biofilms and their disinfection and removal have been important subjects in the maintenance of water quality in areas such as public spas, swimming pools, food processing lines, industrial water systems, and in the hygienic control of medical devices, hospital procedures, etc. Presented here is an outline of biofilm formation, as well as studies on the disinfection and removal of biofilms by oxidizing biocides using established biofilms. These studies using established biofilms may increase the understanding of the variable response of biofilms to planktonic bacteria, and the unique aspects of oxidizing biocides in the disinfection and removal of biofilms.

Impacts of bleach on bryozoans: A framework to distinguish direct and indirect effects using chemical and physical manipulations

Chemical disturbances, caused by contamination, are a global issue and can cause changes in the abundance of populations of one or more species via direct and/or indirect effects. This, in turn, can have profound consequences on assemblages and/or systems. Understanding how contaminants affect functional groups or taxa, i.e. which ecological or biological processes are being affected, is necessary to better predict their consequences. To distinguish between direct and indirect effects of contaminants, however, specific experiments, physically manipulating the changing variable (in this case, biofilm) as well as the contaminant, are necessary. Here, I tested which processes were affected by bleach, a biocide commonly found in urban run-offs that caused a decrease in covers of bryozoans. Effects of bleach on recruitment and growth of bryozoans were variable, suggesting that impacts are complex. Nevertheless, results indicate that bleach reduced recruitment of bryozoans. Therefore, manipulative field experiments were done to test whether these effects were direct or indirect, through changes in the abundance of photosynthetic biofilms. Responses of biofilms varied with the duration and exposure to bleach, as well as the timing of experiments. Abundance of biofilms did not seem, however, to affect the number of recruits of bryozoans, which could suggest a direct effect of bleach on bryozoan recruitment. Given that bryozoans are a major component of subtidal benthic assemblages and an important food source for >300 species, decreases in their abundance, as those observed here, might have important knock-on effects on marine systems due to trophic cascades.Capsule.In situ manipulative experiments showed that bleach decreased covers of bryozoans, which is possibly due to a combination of effects on their growth and recruitment. Manual removals and chemically induced removals are necessary to disentangle direct and indirect effects of contaminants.

Bacterial biofilm under flow: First a physical struggle to stay, then a matter of breathing.

Bacterial communities attached to surfaces under fluid flow represent a widespread lifestyle of the microbial world. Through shear stress generation and molecular transport regulation, hydrodynamics conveys effects that are very different by nature but strongly coupled. To decipher the influence of these levers on bacterial biofilms immersed in moving fluids, we quantitatively and simultaneously investigated physicochemical and biological properties of the biofilm. We designed a millifluidic setup allowing to control hydrodynamic conditions and to monitor biofilm development in real time using microscope imaging. We also conducted a transcriptomic analysis to detect a potential physiological response to hydrodynamics. We discovered that a threshold value of shear stress determined biofilm settlement, with sub-piconewton forces sufficient to prevent biofilm initiation. As a consequence, distinct hydrodynamic conditions, which set spatial distribution of shear stress, promoted distinct colonization patterns with consequences on the growth mode. However, no direct impact of mechanical forces on biofilm growth rate was observed. Consistently, no mechanosensing gene emerged from our differential transcriptomic analysis comparing distinct hydrodynamic conditions. Instead, we found that hydrodynamic molecular transport crucially impacts biofilm growth by controlling oxygen availability. Our results shed light on biofilm response to hydrodynamics and open new avenues to achieve informed design of fluidic setups for investigating, engineering or fighting adherent communities.

Genome amplification and promoter mutation expand the range of csgD-dependent biofilm responses in an STEC population.

Expression of the major biofilm components of E. coli, curli fimbriae and cellulose, requires the CsgD transcription factor. A complex regulatory network allows environmental control of csgD transcription and biofilm formation. However, most clinical serotype O157 : H7 strains contain prophage insertions in the csgD regulator, mlrA, or mutations in other regulators that restrict csgD expression. These barriers can be circumvented by certain compensating mutations that restore higher csgD expression. One mechanism is via csgD promoter mutations that switch sigma factor utilization. Biofilm-forming variants utilizing RpoD rather than RpoS have been identified in glycerol freezer stocks of the non-biofilm-forming food-borne outbreak strain, ATCC 43894. In this study we used whole genome sequencing and RNA-seq to study genotypic and transcriptomic differences between those strains. In addition to defining the consequences of the csgD promoter switch and identifying new csgD-controlled genes, we discovered a region of genome amplification in our laboratory stock of 43894 (designated 43894OW) that contributed to the regulation of csgD-dependent properties.

Novel dental adhesive with triple benefits of calcium phosphate recharge, protein-repellent and antibacterial functions

ObjectiveA new adhesive containing nanoparticles of amorphous calcium phosphate (NACP) with calcium (Ca) and phosphate (P) ion rechargeability was recently developed; however, it was not antibacterial. The objectives of this study were to: (1) develop a novel adhesive with triple benefits of Ca and P ion recharge, protein-repellent and antibacterial functions via dimethylaminohexadecyl methacrylate (DMAHDM) and 2-methacryloyloxyethyl phosphorylcholine (MPC); and (2) investigate dentin bond strength, protein adsorption, Ca and P ion concentration, microcosm biofilm response and pH properties. MethodsMPC, DMAHDM and NACP were mixed into a resin consisting of ethoxylated bisphenol A dimethacrylate (EBPADMA), pyromellitic glycerol dimethacrylate (PMGDM), 2-hydroxyethyl methacrylate (HEMA) and bisphenol A glycidyl dimethacrylate (BisGMA). Protein adsorption was measured using a micro bicinchoninic acid method. A human saliva microcosm biofilm model was tested on resins. Colony-forming units (CFU), live/dead assay, metabolic activity, Ca and P ion concentration and biofilm culture medium pH were determined. ResultsThe adhesive with 5% MPC+5% DMAHDM+30% NACP inhibited biofilm growth, reducing biofilm CFU by 4 log, compared to control (p<0.05). Dentin shear bond strengths were similar (p>0.1). Biofilm medium became a Ca and P ion reservoir having ion concentration increasing with NACP filler level. The adhesive with 5% MPC+5% DMAHDM+30% NACP maintained a safe pH>6, while commercial adhesive had a cariogenic pH of 4. SignificanceThe new adhesive with triple benefits of Ca and P ion recharge, protein-repellent and antibacterial functions substantially reduced biofilm growth, reducing biofilm CFU by 4 orders of magnitude, and yielding a much higher pH than commercial adhesive. This novel adhesive is promising to protect tooth structures from biofilm acids. The method of using NACP, MPC and DMAHDM is promising for application to other dental materials to combat caries.

Subgingival microbiota dysbiosis in systemic lupus erythematosus: association with periodontal status

BackgroundPeriodontitis results from the interaction between a subgingival biofilm and host immune response. Changes in biofilm composition are thought to disrupt homeostasis between the host and subgingival bacteria resulting in periodontal damage. Chronic systemic inflammatory disorders have been shown to affect the subgingival microbiota and clinical periodontal status. However, this relationship has not been examined in subjects with systemic lupus erythematosus (SLE). The objective of our study was to investigate the influence of SLE on the subgingival microbiota and its connection with periodontal disease and SLE activity.MethodsWe evaluated 52 patients with SLE compared to 52 subjects without SLE (control group). Subjects were classified as without periodontitis and with periodontitis. Oral microbiota composition was assessed by amplifying the V4 region of 16S rRNA gene from subgingival dental plaque DNA extracts. These amplicons were examined by Illumina MiSeq sequencing.ResultsSLE patients exhibited higher prevalence of periodontitis which occurred at a younger age compared to subjects of the control group. More severe forms of periodontitis were found in SLE subjects that had higher bacterial loads and decreased microbial diversity. Bacterial species frequently detected in periodontal disease were observed in higher proportions in SLE patients, even in periodontal healthy sites such as Fretibacterium, Prevotella nigrescens, and Selenomonas. Changes in the oral microbiota were linked to increased local inflammation, as demonstrated by higher concentrations of IL-6, IL-17, and IL-33 in SLE patients with periodontitis.ConclusionsSLE is associated with differences in the composition of the microbiota, independently of periodontal status.

Legacy effects and memory loss: how contingencies moderate the response of rocky intertidal biofilms to present and past extreme events.

Understanding how historical processes modulate the response of ecosystems to perturbations is becoming increasingly important. In contrast to the growing interest in projecting biodiversity and ecosystem functioning under future climate scenarios, how legacy effects originating from historical conditions drive change in ecosystems remains largely unexplored. Using experiments in combination with stochastic antecedent modelling, we evaluated how extreme warming, sediment deposition and grazing events modulated the ecological memory of rocky intertidal epilithic microphytobenthos (EMPB). We found memory effects in the non-clustered scenario of disturbance (60days apart), where EMPB biomass fluctuated in time, but not under clustered disturbances (15days apart), where EMPB biomass was consistently low. A massive grazing event impacted on EMPB biomass in a second run of the experiment, also muting ecological memory. Our results provide empirical support to the theoretical expectation that stochastic fluctuations promote ecological memory, but also show that contingencies may lead to memory loss.