Drag force and surface roughness measurements on freshwater biofouled surfaces

The detrimental effect of biofilms on skin friction for near wall flows is well known. The diatom genera Gomphonema and Tabellaria dominated the biofilm mat in the freshwater open channels of the Tarraleah Hydropower Scheme in Tasmania, Australia. A multi-faceted approach was adopted to investigate the drag penalty for biofouled 1.0 m × 0.6 m test plates which incorporated species identification, drag measurement in a recirculating water tunnel and surface characterisation using close-range photogrammetry. Increases in total drag coefficient of up to 99% were measured over clean surface values for biofouled test plates incubated under flow conditions in a hydropower canal. The effective roughness of the biofouled surfaces was found to be larger than the physical roughness; the additional energy dissipation was caused in part by the vibration of the biofilms in three-dimensions under flow conditions. The data indicate that there was a roughly linear relationship between the maximum peak-to-valley height of a biofilm and the total drag coefficient.     

Residual particle free rough surfaces after rough blasting with steel grit in total hip arthroplasty]

INTRODUCTION: Several shot peening and grit blasting techniques are used to modify the surface in the production of hip endoprostheses. A number of publications in maxillo- facial- surgery and orthopedic surgery demonstrated significant contamination on alumina blasted surfaces. Latest research studies suspected an association between surface contamination and early failure of endoprostheses associated with third body wear. Additionally, the European standard EN 12010 requires surfaces free of residual particles. The aim of this study is to evaluate the effects of rough blasting using steel grit followed by a pH dependent cleaning procedures with regard to complying with EN 12010. MATERIAL AND METHOD: Ti6Al7Nb rods were rough blasted 1 time using steel grit ("Stahldrahtkorn", hardness: 56 HRC; R+K Draht GmbH, Leisnig). All blasting procedures were carried out with a special grit blasting device with direct pressure. One Ti6Al7Nb rod each was grit blasted with 3 and 4 bar pressure and 10 rods with 5 bar pressure. Surface roughness was measured by means of Mitutoyo- Surftest 301 three times before and after grit blasting. The mean value was calculated. A field emission scanning electron microscopy (SEM, LEO 1525) was used for the detection of residual particles on the surface of the rods with a backscattered electron detector. RESULTS: By rough blasting using steel grit a mean roughness of 54,9 microm and a maximum roughness of 61,4 microm could be achieved. The surface of all rods showed a considerable contamination with steel particles after grit blasting. After the a pH- dependent cleaning procedure a residual particle free surface was detected in all samples. DISCUSSION: The European standard EN 12010 can be fulfilled using rough blasting with steel grit followed by a pH dependent cleaning procedure.     

Listeria monocytogenes Can Form Biofilms in Tap Water and Enter Into the Viable but Non-Cultivable State

Listeria monocytogenes is a foodborne pathogen that can be transmitted through contaminated raw food or by ready-to-eat products that have been in contact with contaminated surfaces. Tap water (TW) is used to wash produce, as a processed food constituent and to wash processing surfaces and floors. The main aim of this work was to investigate the formation and survival of L. monocytogenes biofilms on stainless steel (SS) coupons in TW at 4, 22, 30 and 37 °C. For that, coupons with biofilm were visualised in situ while other coupons were scraped to quantify total cells by SYTO 9, cultivable numbers by plating onto brain heart infusion agar and viable numbers by the direct viable count method. Results showed that L. monocytogenes can form biofilms on SS surfaces in TW at any temperature, including at 4 °C. The number of total cells was similar for all the conditions tested while cultivable numbers varied between the level of detection (<8.3 CFU cm^?2) and 3.5?×?10⁵ CFU cm^?2, meaning between 7.0?×?10⁴ and 1.1?×?10⁷ cells cm^?2 have entered the viable but non-cultivable (VBNC) state. This work clearly demonstrates that L. monocytogenes can form biofilms in TW and that sessile cells can remain viable and cultivable in some conditions for at least the 48 h investigated. On the other hand, VBNC adaptation suggests that the pathogen can remain undetectable using traditional culture recovery techniques, which may give a false indication of processing surface hygiene status, leading to potential cross-contamination of food products.     

Flowing biofilms as a transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system

Abstract

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 μm h^?1 in the turbulent flow cell and 1.0 μm h^?1 in the laminar flow cell.     

Bacterial Community Structure of Biofilms on Artificial Surfaces in an Estuary

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.     

Synthesis,characterization and in vitro activity of a surface-attached antimicrobial cationic peptide

Infection associated with implanted biomaterials is common and costly and such infections are extremely resistant to antibiotics and host defenses. Consequently, there is a need to develop surfaces which resist bacterial adhesion and colonization. The broad spectrum synthetic cationic peptide melimine has been covalently linked to a surface via two azide linkers, 4-azidobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA), and the resulting surfaces characterized by X-ray photoelectron spectroscopy and contact angle measurements. The quantity of bound peptide was estimated by a modified Bradford assay. The antimicrobial efficacy of the two melimine-modified surfaces against Pseudomonas aeruginosa and Staphylococcus aureus was compared by scanning electron microscopy (SEM) and fluorescence microscopy. Attachment of melimine via ABA gave an approximately 4-fold greater quantity of melimine bound to the surface than attachment via FNA. Surfaces melimine-modified by either attachment strategy showed significantly reduced bacterial adhesion for both strains of bacteria. P. aeruginosa exposed to ABA–melimine and FNA–melimine surfaces showed marked changes in cell morphology when observed by SEM and a reduction of approximately 15-fold (p < 0.001) in the numbers of adherent bacteria compared to controls. For the ABA–melimine surface there was a 33% increase in cells showing damaged membranes (p = 0.0016) while for FNA–melimine there was no significant difference. For S. aureus there were reductions in bacterial adhesion of approximately 40-fold (p < 0.0001) and 5-fold (p = 0.008) for surfaces modified with melimine via ABA or FNA, respectively. There was an increase in cells showing damaged membranes on ABA–melimine surfaces of approximately 87% (p = 0.001) compared to controls, while for FNA–melimine there was no significant difference observed. The data presented in this study show that melimine has excellent potential for development as a broad spectrum antimicrobial coating for biomaterial surfaces. Further, it was observed that the efficacy of antimicrobial activity is related to the method of attachment.     

Gas-phase removal of biofilms from various surfaces using carbon dioxide aerosols

The present study evaluated the removal of Escherichia coli XL1-blue biofilms using periodic jets of carbon dioxide aerosols (a mixture of solid and gaseous CO₂) with nitrogen gas. The aerosols were generated by the adiabatic expansion of high-pressure CO₂ gas through a nozzle and used to remove air-dried biofilms. The areas of the biofilms were measured from scanning electron micrographs before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured with various air-drying times of the biofilms before the treatment, surface materials, and durations of CO₂ aerosols in each 8-s aerosol–nitrogen cleaning cycle. Nearly 100% of the fresh biofilms were removed from the various surfaces very reliably within 90 s. This technique can be useful for removing unsaturated biofilms on solid surfaces and has potential applications for cleaning bio-contaminated surfaces.     

Impacts of hydrodynamic conditions and microscale surface roughness on the critical shear stress to develop and thickness of early-stage Pseudomonas putida biofilms

Biofilms can increase pathogenic contamination of drinking water, cause biofilm-related diseases, alter the sediment erosion rate, and degrade contaminants in wastewater. Compared with mature biofilms, biofilms in the early-stage have been shown to be more susceptible to antimicrobials and easier to remove. Mechanistic understanding of physical factors controlling early-stage biofilm growth is critical to predict and control biofilm development, yet such understanding is currently incomplete. Here, we reveal the impacts of hydrodynamic conditions and microscale surface roughness on the development of early-stage Pseudomonas putida biofilm through a combination of microfluidic experiments, numerical simulations, and fluid mechanics theories. We demonstrate that early-stage biofilm growth is suppressed under high flow conditions and that the local velocity for early-stage P. putida biofilms (growth time < 14 h) to develop is about 50 μm/s, which is similar to P. putida's swimming speed. We further illustrate that microscale surface roughness promotes the growth of early-stage biofilms by increasing the area of the low-flow region. Furthermore, we show that the critical average shear stress, above which early-stage biofilms cease to form, is 0.9 Pa for rough surfaces, three times as large as the value for flat or smooth surfaces (0.3 Pa). The important control of flow conditions and microscale surface roughness on early-stage biofilm development, characterized in this study, will facilitate future predictions and managements of early-stage P. putida biofilm development on the surfaces of drinking water pipelines, bioreactors, and sediments in aquatic environments.     

Impact of diatomaceous biofilms on the frictional drag of fouling-release coatings

Skin-friction results are presented for fouling-release (FR) hull coatings in the unexposed, clean condition and after dynamic exposure to diatomaceous biofilms for 3 and 6 months. The experiments were conducted in a fully developed turbulent channel flow facility spanning a wide Reynolds number range. The results show that the clean FR coatings tested were hydraulically smooth over much of the Reynolds number range. Biofilms, however, resulted in an increase in skin-friction of up to 70%. The roughness functions for the biofilm-covered surfaces did not display universal behavior, but instead varied with the percentage coverage by the biofilm. The effect of the biofilm was observed to scale with its mean thickness and the square root of the percentage coverage. A new effective roughness length scale (k_eff) for biofilms based on these parameters is proposed. Boundary layer similarity-law scaling is used to predict the impact of these biofilms on the required shaft power for a mid-sized naval surface combatant at cruising speed. The increase in power is estimated to be between 1.5% and 10.1% depending on the biofilm thickness and percentage coverage.     

Model system studies of the influence of bacterial biofilm formation on mineral surface reactivity

Biofilm development on mineral surfaces and related changes in surface reactivity were studied using batch and flow through experiments. An artificial groundwater was used as the primary nutrient medium, Pseudomonas aeruginosa (PAO1) was the model microbial organism and ‘mineral’ surfaces were kept as simple as possible by using glass or a polished quartz tile. Experiments were also completed with very low concentrations (100 mg l^?1) of iron, Fe²⁺ , in the solution. In situ confocal laser scanning microscopy of developing colonies during the live growth phase, and of thick, mature biofilms, revealed only sporadic coverage of biofilm cells and associated polymers at the ‘mineral–microbe interface’. Imaging and analysis of biofilm-conditioned surfaces doped with Fe²⁺ -rich solutions allowed the locus and form of Fe-rich mineral precipitation to be determined and show that biological surface components can cause mineral precipitation from dilute dissolved species which might otherwise remain in solution.     

Effectiveness of phages in the decontamination of Listeria monocytogenes adhered to clean stainless steel, stainless steel coated with fish protein, and as a biofilm

Listeria monocytogenes is a food-borne pathogen which causes listeriosis and is difficult to eradicate from seafood processing environments; therefore, more effective control methods need to be developed. This study investigated the effectiveness of three bacteriophages (LiMN4L, LiMN4p and LiMN17), individually or as a three-phage cocktail at ≈9 log₁₀ PFU/ml, in the lysis of three seafood-borne L. monocytogenes strains (19CO9, 19DO3 and 19EO3) adhered to a fish broth layer on stainless steel coupon (FBSSC) and clean stainless steel coupon (SSC), in 7-day biofilm, and dislodged biofilm cells at 15 ± 1 °C. Single phage treatments (LiMN4L, LiMN4p or LiMN17) decreased bacterial cells adhered to FBSSC and SSC by ≈3–4.5 log units. Phage cocktail reduced the cells on both surfaces (≈3.8–4.5 and 4.6–5.4 log₁₀ CFU/cm², respectively), to less than detectable levels after ≈75 min (detection limit = 0.9 log₁₀ CFU/cm²). The phage cocktail at ≈5.8, 6.5 and 7.5 log₁₀ PFU/cm² eliminated Listeria contamination (≈1.5–1.7 log₁₀ CFU/cm²) on SSC in ≈15 min. One-hour phage treatments (LiMN4p, LiMN4L and cocktail) in three consecutive applications resulted in a decrease of 7-day L. monocytogenes biofilms (≈4 log₁₀ CFU/cm²) by ≈2–3 log units. Single phage treatments reduced dislodged biofilm cells of each L. monocytogenes strain by ≈5 log₁₀ CFU/ml in 1 h. The three phages were effective in controlling L. monocytogenes on stainless steel either clean or soiled with fish proteins which is likely to occur in seafood processing environments. Phages were more effective on biofilm cells dislodged from the surface compared with undisturbed biofilm cells. Therefore, for short-term phage treatments of biofilm it should be considered that some disruption of the biofilm cells from the surface prior to phage application will be required.     

Environmental and centrifugal factors influencing the visco-elastic properties of oral biofilms in vitro

Centrifugal compaction causes changes in the surface properties of bacterial cells. It has been shown previously that the surface properties of planktonic cells change with increasing centrifugal compaction. This study aimed to analyze the influences of centrifugal compaction and environmental conditions on the visco-elastic properties of oral biofilms. Biofilms were grown out of a layer of initially adhering streptococci, actinomyces or a combination of these. Different uni-axial deformations were induced on the biofilms and the load relaxations were measured over time. Linear-Regression-Analysis demonstrated that both the centrifugation coefficient for streptococci and induced deformation influenced the percentage relaxation. Centrifugal compaction significantly influenced relaxation only upon compression of the outermost 20% of the biofilm (p < 0.05), whereas biofilm composition became influential when 50% deformation was induced, invoking re-arrangement of the bacteria in deeper biofilm structures. In summary, the effects of centrifugal compaction of initially adhering, centrifuged bacteria extend to the visco-elastic properties of biofilms, indicating that the initial bacterial layer influences the structure of the entire biofilm.     

Biofilm community structure and the associated drag penalties of a groomed fouling release ship hull coating

Grooming is a proactive method to keep a ship’s hull free of fouling. This approach uses a frequent and gentle wiping of the hull surface to prevent the recruitment of fouling organisms. A study was designed to compare the community composition and the drag associated with biofilms formed on a groomed and ungroomed fouling release coating. The groomed biofilms were dominated by members of the Gammaproteobacteria and Alphaproteobacteria as well the diatoms Navicula, Gomphonemopsis, Cocconeis, and Amphora. Ungroomed biofilms were characterized by Phyllobacteriaceae, Xenococcaceae, Rhodobacteraceae, and the pennate diatoms Cyclophora, Cocconeis, and Amphora. The drag forces associated with a groomed biofilm (0.75 ± 0.09 N) were significantly less than the ungroomed biofilm (1.09 ± 0.06 N). Knowledge gained from this study has helped the design of additional testing which will improve grooming tool design, minimizing the growth of biofilms and thus lowering the frictional drag forces associated with groomed surfaces.     

A Comparative Study of the Non-acidic Chemically Mediated Antifoulant Properties of Three Sympatric Species of Ascidians Associated with Seagrass Habitats

In this study, the mechanical properties of biofilms formed at the surface of nano-filtration (NF) membranes from a drinking water plant were analysed. Confocal laser scanning microscopy observations revealed that the NF biofilms formed a dense and heterogeneous structure at the membrane surface, with a mean thickness of 32.5 ± 17.7 μm. The biofilms were scraped from the membrane surface and analysed in rotation and oscillation experiments with a RheoStress 150 rotating disk rheometer. During rotation analyses, a viscosity decrease with speed of shearing characteristic of rheofluidification was observed (η = 300 Pa s for ý = 0.3 s^?1). In the oscillation analyses with a sweeping of frequency (1–100 Hz), elasticity (G′) ranged from 3000 to 3500 Pa and viscosity (G″) from 800 to 1200 Pa. Creep curves obtained with an application of a shear stress of 30 Pa were viscoelastic in nature. The G ₀ and η values were, respectively, 1.4 ± 0.3 × 10³ Pa and 3.3 ± 0.65 × 10⁶ Pa s. The relationship between the characteristics of NF biofilms and the flow conditions encountered during NF is discussed.     

Differences in colonisation of five marine bacteria on two types of glass surfaces

The retention patterns of five taxonomically different marine bacteria after attachment on two types of glass surfaces, as-received and chemically etched, have been investigated. Contact angle measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), X-ray fluorescence spectroscopy (XRF) and X-ray photoelectron spectrometry (XPS) were employed to investigate the impact of nanometer scale surface roughness on bacterial attachment. Chemical modification of glass surfaces resulted in a ～1 nm decrease in the average surface roughness (R _a) and the root-mean-squared roughness (R_q ) and in a ～8 nm decrease in the surface height and the peak-to-peak (R _max) and the 10-point average roughness (R_z ). The study revealed amplified bacterial attachment on the chemically etched, nano-smoother glass surfaces. This was a consistent response, notwithstanding the taxonomic affiliation of the selected bacteria. Enhanced bacterial attachment was accompanied by elevated levels of secreted extracellular polymeric substances (EPS). An expected correlation between cell surface wettability and the density of the bacterial attachment on both types of glass surfaces was also reported, while no correlation could be established between cell surface charge and the bacterial retention pattern.     

Plant-derived compounds as natural antimicrobials to control paper mill biofilms

Biofilms can cause severe problems in industrial paper mills, particularly of economic and technological types (clogging of filters, sheet breaks or holes in the paper, machine breakdowns, etc.). We present here some promising results on the use of essential oil compounds to control these biofilms. Biofilms were grown on stainless-steel coupons with a microbial white water consortium sampled from an industrial paper mill. Five essential oil compounds were screened initially in the laboratory in terms of their antimicrobial activity against planktonic cells and biofilms. The three most active compounds were selected and then tested in different combinations. The combination finally selected was tested at the pilot scale to confirm its efficiency under realistic conditions. All the compounds tested were as active against biofilms as they were against planktonic cells. The most active compounds were thymol, carvacrol, and eugenol, and the most efficient combination was thymol–carvacrol. At a pilot scale, with six injections a day, 10 mM carvacrol alone prevented biocontamination for at least 10 days, and a 1 mM thymol–carvacrol combination enabled a 67 % reduction in biofilm dry matter after 11 days. The use of green antimicrobials could constitute a very promising alternative or supplement to the treatments currently applied to limit biofilm formation in the environment of paper mill machines.     

Dynamic surface deformation of silicone elastomers for management of marine biofouling: laboratory and field studies using pneumatic actuation

Many strategies have been developed to improve the fouling release (FR) performance of silicone coatings. However, biofilms inevitably build on these surfaces over time. Previous studies have shown that intentional deformation of silicone elastomers can be employed to detach biofouling species. In this study, inspired by the methods used in soft-robotic systems, controlled deformation of silicone elastomers via pneumatic actuation was employed to detach adherent biofilms. Using programmed surface deformation, it was possible to release > 90% of biofilm from surfaces in both laboratory and field environments. A higher substratum strain was required to remove biofilms accumulated in the field environment as compared with laboratory-grown biofilms. Further, the study indicated that substratum modulus influences the strain needed to de-bond biofilms. Surface deformation-based approaches have potential for use in the management of biofouling in a number of technological areas, including in niche applications where pneumatic actuation of surface deformation is feasible.     

Growth and morphological development of laboratory-reared larvae and juveniles of the Laotioan indigenous cyprinid Hypsibarbus malcolmi

The morphological development, including the pigmentation, body proportions, fins, and survival rate for 30 days after hatching, of laboratory-reared larval and juvenile Hypsibarbus malcolmi is described. Body lengths (BL) of larvae and juveniles were 2.0 ± 0.2 (mean ± SD) mm at 1 h after hatching (day 0) and 9.2 ± 0.6 mm on day 16, reaching 12.1 ± 0.9 mm on day 30. Yolk volume decreased linearly, with the yolk being completely absorbed by day 3 in all preflexion larvae (all specimens >3.2 mm BL). Feeding was observed on day 2 in fish which had rapidly undergone complete yolk absorption following mouth and anus opening on day 1, and on day 3 in all remaining fish. Myomere numbers were 20–21 + 11–12 = 31–33, although they were not clearly visible in juveniles. Melanophores were few on the body during days 0–2, but increased with growth and covered the entire upper dorsal body surface during the juvenile stage. Body proportions tended to become constant in juveniles. Notochord flexion began in larvae >5.2 mm BL on day 8, and was completed in larvae >8.4 mm BL on day 14. Specimens with full fin ray complements were initially observed on day 22 (10.4 mm BL in juveniles). All specimens >11.5 mm BL had attained the juvenile stage. A high survival rate of 92.7% was estimated on day 30.     

Use of an in vitro flat-bed biofilm model to measure biologically active anti-odour compounds

The objective of this study was to demonstrate the utility of a modified flat-bed perfusion biofilm matrix system for testing toothpaste formulations directly, without dilution, as a layer in direct contact with the biofilm matrix surface. Final biofilm yields and volatile sulphur compounds (VSC) biogenesis were measured to show the relative efficacy of toothpaste formulations. Diffusion characteristics of the flat-bed system to exposure with Meridol® tooth and tongue gel (TTG; 1,400 ppm F^? from amine fluoride/stannous fluoride, 0.5 % zinc lactate, oral malodour counteractives) was assessed using a bioluminescent target species Escherichia coli Nissle 1917/pGLITE coupled with a low-light photon camera to visualise the kill kinetics. Tongue-flora derived, mixed culture biofilms (n?=?4) received 5, 15 and 30 min treatment with TTG, respectively, to determine the optimum time of exposure. VSC biogenesis was measured from headspace samples by gas chromatography prior to and following treatment of two daily applications for 4 days of treatment (TTG), positive control (CHX gel) and negative controls (placebo and sham treatment). Viable counts were performed at the end of experiments by destructive sampling of the biofilms and plating onto selective and non-selective agar. Following a single treatment with TTG, the E. coli biofilm with lux target gave >50 % reduction of luminescence within 2 to 3 h before recovering to a steady state over 10 h, suggesting biofilm cidal activity rather biostasis. For mixed culture biofilms, 15- and 30-min treatment exposure with TTG gave almost identical reductions in final biofilm yields. For comparing efficacy of treatments, biofilms treated with TTG gave greatest reductions in both pre–post levels of H₂S (P?<?0.01) and CH₃SH (P?<?0.05) and population yields at the end of the experiments (P?<?0.001) compared to placebo and positive control. The in vitro flat-bed perfusion model may be used to replicate many of the activities and reactions believed to be occurring by the tongue biofilm microflora within a real mouth, including VSC biogenesis and its inhibition by exposure to active agents as components of toothpastes and gels applied in direct contact with the biofilm.     

Different methods and storage duration affect measurements of epilithic extracellular enzyme activities in lotic biofilms

Extracellular enzyme activity (EEA) is becoming increasingly common for measuring biofilm function in streams. Different methods for enzyme assays may yield results that cannot be compared among studies, and duration of sample storage may also affect EEAs, leading to erroneous conclusions. We compared two frequently used methods for measuring phosphatase (PHOS), leucine aminopeptidase (LAMP), β-glucosidase (GLU), and β-xylosidase (XYLO) by conducting assays with intact and disrupted epilithic biofilms grown on tiles in three streams. Storage duration effects on EEA were documented with intact and disrupted biofilms kept in the dark at 4°C for 3 and 5 days. Intact biofilms had significantly less EEA than disrupted biofilms for all enzymes (P < 0.01). The two methods gave conflicting EEA results among streams, and ratios of disrupted to intact EEAs for each enzyme were not consistent among the three streams. PHOS was significantly greater than day 1 measurements when stored as disrupted (Day 3 = 210%, Day 5 = 199% increases) and intact biofilms (Day 3 = 375%, Day 5 = 240% increases). LAMP activities were significantly less when stored as disrupted biofilms (Day 3 = ?49% decrease) and greater when stored as intact biofilms (Day 5 = 72% increase). GLU (Day 3 = 313% increase) and XYLO (Day 3 = 121%, Day 5 = 188% increases) were significantly greater when stored as intact biofilms. The magnitude of change for all EEAs was inconsistent among streams, indicating that a consistent correction factor cannot be used to account for variation associated with storage duration. Consistent methods must be used and storage time should be minimized, preferably to the day of sampling, for valid inter-study comparisons. Conclusions can significantly differ between the two methods, therefore having implications for inter-study comparisons, understanding of biofilm function and dynamics, and environmental management decisions.