Surface-grafted, environmentally sensitive polymers for biofilm release

Controlling bacterial biofouling is desirable for almost every human enterprise in which solid surfaces are introduced into nonsterile aqueous environments. One approach that is used to decrease contamination of manufactured devices by microorganisms is using materials that easily slough off accumulated material (i.e., fouling release surfaces). The compounds currently used for this purpose rely on low surface energy to inhibit strong attachment of organisms. In this study, we examined the possible use of environmentally responsive (or "smart") polymers as a new class of fouling release agents; a surface-grafted thermally responsive polymer, poly(N-isopropylacrylamide) (PNIPAAM), was used as a model compound. PNIPAAM is known to have a lower critical solubility temperature of approximately 32 degrees C (i.e., it is insoluble in water at temperatures above 32 degrees C and is soluble at temperatures below 32 degrees C). Under experimental conditions, >90% of cultured microorganisms (Staphylococcus epidermidis, Halomonas marina) and naturally occurring marine microorganisms that attached to grafted PNIPAAM surfaces during 2-, 18-, 36-, and 72-h incubations were removed when the hydration state of the polymer was changed from a wettability that was favorable for attachment to a wettability that was less favorable. Of particular significance is the observation that an organism known to attach in the greatest numbers to hydrophobic substrata (i.e., H. marina) was removed when transition of PNIPAAM to a more hydrated state occurred, whereas an organism that attaches in the greatest numbers to hydrophilic substrata (i.e., S. epidermidis) was removed when the opposite transition occurred. Neither solvated nor desolvated PNIPAAM exhibited intrinsic fouling release properties, indicating that the phase transition was the important factor in removal of organisms. Based on our observations of the behavior of this model system, we suggest that environmentally responsive polymers represent a new approach for controlling biofouling release.     

Thermocontrol of solute permeation across polymer membrane composed of poly(N,N-dimethylaminoethyl methacrylate) and its copolymers

Polymer membranes composed ofN,N-dimethylaminoethyl methacrylate (DMAEMA) and acrylamide (AAm) (or ethyl acrylamide (EAAm)) were prepared to demonstrate the thermocontrol of solute permeation. Poly DMEMA has a lower critical solution temperature (LCST) at around 50°C in water. With the copolymerization of DMAEMA with AAm (or EAAm), a shift in the LCST to a lower temperature was observed, probably due to the formation of hydrogen bonds between the amide andN,N-dimethylamino groups. However, the temperature-induced phase transition of poly (DMAEMA-co-EAAm) did not show a similar trend to that of poly (DMAEMA-co-AAm) in the gel state. The hydrogen bonds in poly (DMAEMA-co-EAAm) were significantly disrupted with the formation of a gel network, which led to a difference in the swelling behavior of polymer gels in response to temperature. To apply these polymers to temperature-sensitive solute permeation, polymer membranes were prepared. The permeation pattern of hydrocortisone, used as the model solute, was explained based on the temperature-sensitive swelling behavior of the polymer membranes.     

Prediction of lower critical solution temperature of N-isopropylacrylamide–acrylic acid copolymer by an artificial neural network model

In this paper, we have investigated the lower critical solution temperature (LCST) of N-isopropylacrylamide–acrylic acid (NIPAAm-AAc) copolymer as a function of chain-transfer agent/initiator mole ratio, acrylic acid content of copolymer, concentration, pH and ionic strength of aqueous copolymer solution. Aqueous solutions with the desired properties were prepared from previously purified polymers, synthesized at 65 °C by solution polymerization using ethanol. The effects of each parameter on the LCST were examined experimentally.In addition, an artificial neural network model that is able to predict the lower cretical solution temperature was develeped. The predictions from this model compare well against both training and test data sets with an average error less than 2.53%.Figure Cross plot of predicted and experimental LCST values for the testing data set.     

Engineering temperature-sensitive poly(N-isopropylacrylamide) polymers as carriers of therapeutic proteins

This study was carried out to engineer N-isopropylacrylamide (NiPAM) polymers that contain protein-reactive N-acryloxysuccinimide (NASI) and hydrophobic alkylmethacrylates (AMAs). These thermoreversible, protein-conjugating polymers hold potential for retention of therapeutic proteins at an application site where tissue regeneration is desired. The lower critical solution temperatures (LCST) of the polymers were effectively controlled by the AMA mole content. The AMAs with longer side-chains were more effective in lowering the LCST. Polymers without NASI exhibited a stable LCST in phosphate buffer and in serum over a 10-day study period. The LCST of polymers containing NASI was found to increase over time in phosphate buffer, but not in serum-containing medium. The LCST increase in phosphate buffer was proportional to the AMA content. The feasibility of localizing a therapeutic protein, recombinant human bone morphogenetic protein-2 (rhBMP-2), to a site of application was explored in a rat intramuscular injection model. The results indicated that polymers capable of conjugating to rhBMP-2 were most effective in localizing the protein irrespective of the LCST (13-25 degrees C). For polymers with no NASI groups, a lower LCST resulted in a better rhBMP-2 localization. We conclude that thermosensitive polymers can be engineered for delivery of therapeutic proteins to improve their therapeutic efficacy.     

Salinity effects on the leaf water relations components and ion accumulation patterns in Avicennia germinans (L.) L. seedlings

Physiological traits involved in leaf water relations were evaluated in Avicennia germinans (L.) L. seedlings growing at different salinities in the field. Analysis of pressure-volume (P-V) curves and sap osmometry were combined to evaluate osmotic adjustment and cell elasticity, and the contribution of accumulated inorganic ions to osmotic potential was estimated. Seedlings growing in soils with interstitial water salinity above that of normal sea water showed a modification of the relationship between water potential and relative water content. Thus, their leaf osmotic potential at maximum turgor (Ψ_{π( max )}) and at zero turgor (Ψ_π(0)) was 1.41 and 1.82 MPa lower respectively, than that of the seedlings from the low salinity site. Volumetric moduli of elasticity () were between 17 and 23 MPa. Thus, ɛ was about 6 MPa lower in high-salinity plants indicating that their cells were slightly more elastic. Ionic concentration analysis showed that Σ [anions] and Σ [cations] were higher in the high-salinity site (22–35%) while the water content per unit dry mass was only 12–17% lower. Reduction in water content was insufficient to explain the increase in ion concentration. Ion concentration explained 73 and 66% of the osmotic potential estimated by P-V curves for leaves from low- and high-salinity sites, respectively. In conclusion, this study provided evidence that leaves of A. germinans seedlings adapt to hypersaline soils by increasing solute concentration by 52% and cell elasticity by 26%. Both processes allow leaf water uptake and turgor maintenance over a large range of soil water potential. Received: 30 June 1997 / Accepted 26 November 1997     

Occurrence of the benthic trachymedusa Ptychogastria polaris Allman, 1878 (Cnidaria: Hydrozoa) off Northeast Greenland and in the northern Barents Sea

The trachymedusa Ptychogastria polaris Allman, 1878 has been identified in seabed photographs from high-Arctic shelves and upper continental slopes off Northeast Greenland and in the northern Barents Sea. It was found to be a common epifaunal element, being present at 34 of 57 stations in 40- to 495-m depth and at 7 of 11 stations in 70- to 330-m depth, respectively. All specimens recorded in the photographs either sat directly at, or hovered very closely (≤ about 1 cm) over, the sea bed, indicating a primarily epibenthic life style of this hydrozoan species. The small-scale (i.e. within-station) distribution of medusae was rather patchy, with frequencies along photographic transects – consisting of 35–73 pictures distributed over seabed strips of 150- to 300-m length – varying from 1 to 58% off Northeast Greenland and from 1 to 34% in the Barents Sea. Absolute maximum density was 6 ind m⁻², and station mean abundances ranged from 0.01 to 0.91 ind m⁻² and from 0.01 to 0.52 ind m⁻², respectively. Values tended to decrease with water depth, albeit significantly only off Northeast Greenland. Otherwise, no clear relationships to environmental conditions, such as geomorphology or near-bottom water hydrography, were detected. Circumstantial evidence suggests that seabed granulometry and potential food supply are important distribution determinants. However, further investigations are required to identify more stringently the key factors controlling the distribution of P. polaris. Received: 8 September 1997 / Accepted: 12 September 1997     

Photosynthesis and water relations of four oak species: impact of flooding and salinity

 As global climate changes, sea level rise and increased frequency of hurricanes will expose coastal forests to increased flooding and salinity. Quercus species are frequently dominant in these forest, yet little is known about their salinity tolerance, especially in combination with flooding. In this study, 1-year-old seedlings of Quercus lyrata Walt. (overcup oak), Q. michauxii Nutt. (swamp chestnut oak), Q. nigra L. (water oak), and Q. nuttallii Palmer (Nuttall oak) were chronically (simulating sea level rise) and acutely (simulating hurricane storm surge) exposed to increased flooding and salinity, individually and in combination. The four species demonstrated two response patterns of photosynthesis (A), conductance, and leaf water potential, apparently related to their relative flood tolerance. In Q. lyrata, Q. nuttallii, and Q. nigra (moderately flood-tolerant), A was not immediately reduced after the initiation of the freshwater flooding, but was reduced as the duration of flooding increased. In the second pattern, demonstrated by the weakly flood-tolerant Q. michauxii, A was immediately reduced by freshwater flooding with an increasing impact over time. Watering with 2 parts per thousand (ppt) saline water did not consistently reduce A, but flooding with 2 ppt reduced A of all species, similar to the response with freshwater flooding. Photosynthesis of all species was reduced by 6 ppt watering or flooding, with the latter treatment killing all species within 8 weeks. When acutely exposed to 30 ppt salinity, A was quickly and severely reduced regardless of whether the seedlings were watered or flooded. Acutely flooded seedlings exposed to high salinity died within 2 weeks, but seedlings watered with 30 ppt saline water recovered and A was not reduced the following spring. As saline flooding of coastal areas increases due to sea level rise, photosynthesis of these species will be differentially affected based primarily on their flood tolerance. This suggests that increased flooding associated with sea level rise will impact these tree species to a greater extent than small increases in soil salinity. High salinity accompanying storm surges will be very harmful to all of these species. Received: 20 October 1997 / Accepted: 2 December 1998     

Production of xanthan gum and ice-nucleating material from whey by Xanthomonas campestris pv. translucens

Xanthomonas campestris pv. translucens IFO13599 could produce xanthan gum (18.5 mg/100 mg, lactose) with lactose as the growth substrate in spite of a low level of β-galactosidase. This productivity corresponded to one-fifth that with glucose. This strain could also produce ice-nucleating material having an ice-nucleating temperature, T ₅₀, of −2.8 °C with xanthan gum in the culture broth. We found that this strain produced both materials in whey medium from which the insoluble components had been removed. The production of xanthan with ice-nucleating material reached a maximum after cultivation for 168 h under optimum conditions. Furthermore, the xanthan obtained had a low viscosity because of its variant structure revealed, by TLC and HPLC analyses, to be lacking pyruvic acid. Furthermore, we concluded that this mixture had considerable potential as a regeneratic agent, when compared to other regeneratic agents such as carboxymethylcellulose. Received: 29 August 1997 / Received revision: 17 November 1997 / Accepted: 18 November 1997     

Morphofunctional changes of the interrenal gland and of ultrastructure of chloride cells in intact and hypophysectomized juveniles of starred sturgeon <Emphasis Type="Italic">Acipenser stellatus</Emphasis> (Acipenseridae) in the course of adaptation to sea water

The impact of hypophysectomy on the state of the interrenal gland and ultrastructure of chloride cells of gills is investigated in 18-month old juveniles of starred sturgeon Acipenser stellatus in the process of its adaptation to artificial sea water (14.6‰). Hypophysectomized juveniles, similarly to intact juveniles, are able to support a relative stability of osmolarity of blood serum in the course of adaptation to sea water by transition from hyperosmotic to hypoosmotic type of osmoregulation. Changes in the investigated parameters of cells of the interrenal gland (volume of nuclei, areas of cells and of lipophilic vacuoles) occurring in the hypophysectomized and in intact specimens in the process of adaptation to sea water are generally similar, but have different dynamics. In contrast to many teleostean species, in acipenserids the hypophyectomy does not cause atrophy of the interrenal gland. The latter is incorporated in the process of regulation in the course of adaptation of fish to sea water. Hypophyecotmy results in partial destruction of organoids in some chloride cells of gills. However, when the fish are transferred to sea water, the structural changes occur in chloride cells characteristic of their transition to the excretory state. This may happen only at activation of the transport enzyme Na⁺/K⁺-ATPase of these cells by cortisol produced by the interrenal gland. In the absence of hypophysis, the functional connection of organs of the axis hypothalamus (ACTH-immunopositive cells of tuberal nucleus) → the interrenal gland → chloride cells is realized in the fish.     

Comparison of attached algal communities on natural and artificial substrata along a thermal gradient

Colonization rate and community structure of periphyton assemblages was examined on aluminium and glass substrata and compared to populations on four submerged macrophyte species in three temperature zones in Cholla Lake, Arizona, U.S.A. Higher densities were achieved over shorter incubation intervals in the warmer zones (26–35° C). Representatives from the planktonic diatom community were first to colonize artificial substrata during the initial two hour incubation period in all temperature zones. Two periphyton diatom representatives, Amphora coffeiformis and Cocconeis placentula var. lineata were the numerical dominants after one week. Cocconeis placentula var. lineata was most competitive on natural substrata at temperatures <26°C, while Amphora coffeiformis dominated temperature zones >26°C with no significant preferences for artificial or natural substrata. The significance of temperature, specific conductance and availability of living hosts is discussed with respect to regulating populations of these two common periphytic diatom species in alkaline waters in southwestern U.S.A. Similarity indices (SIMI) were used to compare algal assemblages on various natural and artificial substrata pairs. Periphyton assemblages were very similar on all natural substrata within similar temperature zones, with little or no preference for macrophyte species displaying similar leaf morphology. Diatom assemblages were quite similar on aluminium and glass substrata throughout the incubation period in all temperature zones, while blue-green algal populations were significantly different, particularly in the higher temperature zones (>28°C). Natural periphyton communities were best represented after four weeks incubation with aluminium substrata in warmer temperature zones (>28°C) or where filamentous blue-green algae dominated. The selection of adequate incubation time when employing artificial substrata to evaluate natural assemblages for different environmental conditions and algal populations is discussed.     

In situ harvesting of adhered target cells using thermoresponsive substrate under a microscope: principle and instrumentation

A novel technique and instrumented device were developed to harvest target cells from multicellular mixture of different cell types under a microscope. The principle of the technique is that cells cultured on a thermoresponsive-substance-coated dish were detached by a region-specific cooling device and simultaneously harvested using a micropipette, both of which were assembled in an inverted microscope. Thermoresponsive coating consists of the mixture of poly(N-isopropylacrylamide) (PNIPAAm) and PNIPAAm-grafted gelatin. The former non-cell-adhesive polymer dissolves below at 32.1 degrees C in water and precipitates over that temperature (called lower critical solution temperature, LCST), and the latter cell-adhesive polymer has LCST of 34.1 degrees C. The appropriate mixing ratio of these thermoresponsive polymers exhibited high cell adhesion at physiological temperature and complete cell detachment at room temperature. A device developed as to cool at only a tiny area of the bottom of the dish, beneath which a cell that was targeted under a microscope, was assembled in a microscope. It was demonstrated that single cell or two cells that adhered to each other was detached from the surface and harvested by a micropipette within approximately 30s.     

Regulation of body temperature in the white shark, Carcharodon carcharias

Stomach temperatures of three white sharks, Carcharodoncarcharias, (one reported previously and two new individuals) were intermittently recorded by acoustic telemetry at the South Farallon Islands, central California. Temperature profiles of the water column were obtained concurrently. Stomach temperatures were elevated over ambient water temperatures by as much as 14.3 °C. Stomach temperatures varied within a narrow range while ambient water temperature fluctuated over a much larger range, showing that this species regulates its body temperature. These data, in combination with previous work on the physiology and anatomy of white sharks, indicate that the white shark is endothermic. It appears that the heat retention system in lamnid sharks has allowed them to inhabit cold water and remain active predators of swift and agile prey. Accepted: 17 February 1997     

Regulation of growth and adhesion of cultured cells by insulin conjugated with thermoresponsive polymers

We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1-10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 339-344, 1997.     

Algal antifouling and fouling-release properties of metal surfaces coated with a polymer inspired by marine mussels

The marine antifouling and fouling-release performance of titanium surfaces coated with a bio-inspired polymer was investigated. The polymer consisted of methoxy-terminated poly(ethylene glycol) (mPEG) conjugated to the adhesive amino acid l-3,4-dihydroxyphenylalanine (DOPA) and was chosen based on its successful resistance to protein and mammalian cell fouling. Biofouling assays for the settlement and release of the diatom Navicula perminuta and settlement, growth and release of zoospores and sporelings (young plants) of the green alga Ulva linza were carried out. Results were compared to glass, a poly(dimethylsiloxane) elastomer (Silastic T2) and uncoated Ti. The mPEG-DOPA3 modified Ti surfaces exhibited a substantial decrease in attachment of both cells of N. perminuta and zoospores of U. linza as well as the highest detachment of attached cells under flow compared to control surfaces. The superior performance of this polymer over a standard silicone fouling-release coating in diatom assays and approximately equivalent performance in zoospore assays suggests that this bio-inspired polymer may be effective in marine antifouling and fouling-release applications.     

Effects of ultraviolet radiation on early larval stages of the Alpine newt, Triturus alpestris, under natural and laboratory conditions

Although Alpine newts (Triturus alpestris) are found at altitudes up to 2500 m, their larvae proved to be extremely sensitive to UV radiation when exposed in clear tapwater to natural sunlight or to comparable artificial UV-B radiation in the laboratory. The experiments revealed severe skin damages (lysis of epithelial cells) and mortality after a few days of exposure. In their natural habitats above the timberline, however, the larvae are protected by the high concentration of dissolved organic carbon in the water leading to an almost complete absorption of UV radiation within the first few centimeters of the water layer. Furthermore, when exposed to UV radiation larvae show erratic swimming activities that may carry them into protected areas. Only in very shallow natural habitats did we detect sublethal UV-like histological effects. Shallow high mountain ponds with clear water normally lack newt populations, probably as a consequence of both low temperature and the effects of UV radiation. Received: 4 October 1996 / Accepted: 7 February 1997     

The influence of climate on durum wheat quality in Tuscany,Central Italy

Climate and meteorological conditions greatly affect agricultural activities, modifying plant responses and determining the quantity and the quality of production. In this respect, the aim of this research was to analyze the quality of winter durum wheat (Triticum turgidum L. var. durum), in terms of protein content through the use of meteorological information. Meteorological conditions were described utilizing both local weather station data (air temperature, cumulated precipitation) and large-scale information available freely on the internet, such as geopotential height (GPH), sea surface temperature (SST), and the North Atlantic Oscillation (NAO) index. The analysis was carried out for the period 1997–2009 in the Tuscany region, Central Italy. Grain protein was positively correlated with air temperature during the February to June period, and negatively with cumulative precipitation during the entire period from November to June. Protein content was also negatively correlated with 500 hPa GPH over Gibraltar and North-Western Africa during the March to June period and with the SST of the Atlantic Ocean south-west of the Canary Islands during the January to June period. Finally, with regard to the NAO, winter durum wheat quality was positively correlated with the specific index for several months, in particular during the winter period. These results demonstrate that precipitation and air temperature over the production area represent two crucial variables driving the vegeto-productive responses of winter durum wheat. On the other hand, the use of large-scale meteorological information showed great potential from the perspective of a local quality forecast system setup.     

Interrelations between respiration and dormancy in buds of three hardwood species with different chilling requirements for dormancy release

 Respiration in vegetative buds of mature Betula pendula, Alnus glutinosa and Prunus padus trees was measured monthly at 15°C from mid-October 1996 to natural outdoor budburst in April 1997. In B. pendula the effect of bud water content on respiration was also estimated (December–April) by artificial imbibition of buds for 24 h prior to measurement of respiration. For estimation of corresponding bud dormancy status, batches of twigs were forced at identical monthly intervals at 15°C in long days (24 h), and budburst recorded. In all species dormancy was deepest when the leaves were shed in October, and dormancy was first alleviated in P. padus followed by B. pendula and A. glutinosa. However, bud respiration capacity was not related to dormancy release as it decreased in all species from October to November and displayed no notable increase until February in P. padus, March in B. pendula and April in A. glutinosa, after completion of dormancy release. Rather, increase in respiration coincided with growth resumption prior to budburst. Artificial imbibition of B. pendula buds increased the water content by approximately 10% (FW) and induced a doubling of the respiration rate (December–February). Moreover, the seasonal variation in bud water content (October–April) explained 94% of the variation in respiration in B. pendula and P. padus, and 84% in A. glutinosa. These observations suggest an important role of water content for respiration. During a cold period from mid-December to mid-January with mean temperature of –9.7°C dormancy release was arrested in P. padus, and to some degree in A. glutinosa, whereas dormancy release progressed normally in B. pendula. This indicates species differences in lower critical temperatures for dormancy release. Received: 30 June 1997 / Acceped: 1 October 1997     

The development of marine biofilms on two commercial non-biocidal coatings: a comparison between silicone and fluoropolymer technologies

The antimicrobial performance of two fouling-release coating systems, Intersleek 700® (IS700; silicone technology), Intersleek 900® (IS900; fluoropolymer technology) and a tie coat (TC, control surface) was investigated in a short term (10 days) field experiment conducted at a depth of ca 0.5 m in the Marina Bandar Rawdha (Muscat, Oman). Microfouling on coated glass slides was analyzed using epifluorescence microscopy and adenosine-5′-triphosphate (ATP) luminometry. All the coatings developed biofilms composed of heterotrophic bacteria, cyanobacteria, seven species of diatoms (2 species of Navicula, Cylindrotheca sp., Nitzschia sp., Amphora sp., Diploneis sp., and Bacillaria sp.) and algal spores (Ulva sp.). IS900 had significantly thinner biofilms with fewer diatom species, no algal spores and the least number of bacteria in comparison with IS700 and the TC. The ATP readings did not correspond to the numbers of bacteria and diatoms in the biofilms. The density of diatoms was negatively correlated with the density of the bacteria in biofilms on the IS900 coating, and, conversely, diatom density was positively correlated in biofilms on the TC. The higher antifouling efficacy of IS900 over IS700 may lead to lower roughness and thus lower fuel consumption for those vessels that utilise the IS900 fouling-release coating.     

Further observations on flagellates within sea ice in northern Bothnian Bay, the Baltic Sea

To improve our knowledge of flagellates inhabiting the Baltic Sea ice and water column during the winter, material was obtained from northern Bothnian Bay in March/April 1995. Light microscopical observations on live and fixed material and further transmission electron microscopy of whole mounts revealed 47 nanoflagellate taxa. In addition, detached scales of eight taxa were encountered. It is now evident that nearly all nanoflagellate classes are present within Bothnian Bay sea ice. The most common groups were cryptomonads, dinoflagellates, chrysophytes, prasinophytes, choanoflagellates and heterotrophic flagellates of unknown systematic position (Protista incertae sedis). Most flagellates in Baltic Sea ice biota apparently thrive in both the water column and the sea ice, while some, e.g. Paraphysomonas spp. (Chrysophyceae), heterotrophic euglenids, volvocalean chlorophytes, and some taxa of uncertain systematic affinity, are more frequently found within the sea ice. Received: 9 February 1997 / Accepted: 23 September 1997     

Impact of biological factors on the ennoblement of stainless steel in Baltic seawater

Open circuit potentials of stainless steels increased when immersed in the Baltic Sea. The ennoblement potential was +200 mV_sce in 40 to 50 days when sea water temperature was below 52°C and +300–400 mV_sce within <40 days at around 102°C. Ennoblement occurred in a laboratory ecosystem at 232°C in 20 to 30 days, and at 262°C in <20 days, but no ennoblement occurred at A322°C within 40 days. By the time the ennoblement was complete, compact microcolonies covered 1–10% of the steel surface. Nutrient enrichment of Baltic Sea water by twofold above the natural levels increased microbial growth but attenuated open circuit potential increase of the stainless steels. Exposure of the ennobled stainless steels to similar levels of nutrients did not reverse the already developed open circuit potentials. Attenuation of the ennobling response of the stainless steels by increases of temperature and eutrophication suggests a role for microorganisms which is crucial for the electrochemical behaviour of steels in brackish Baltic Sea water. Journal of Industrial Microbiology & Biotechnology (2000) 24, 410–420. Received 02 November 1999/ Accepted in revised form 24 March 2000