The effect of turbulent flow and surface roughness on biofilm formation in drinking water

There is considerable interest in both Europe and the USA in the effects of microbiological fouling on stainless steels in potable water. However, little is known about the formation and effects of biofilms, on stainless steel in potable water environments, particularly in turbulent flow regimes. Results are presented on the development of biofilms on stainless steel grades 304 and 316 after exposure to potable water at velocities of 0.32, 0.96 and 1.75 m s⁻¹. Cell counts on slides of stainless steel grades 304 and 316 with both 2B (smooth) and 2D (rough) finishes showed viable and total cell counts were higher at the higher flow rates of 0.96 and 1.75 m s⁻¹, compared to a flow rate of 0.32 m s⁻¹. Extracellular polysaccharide levels were not significantly different (P< 0.05) between each flow rate on all stainless steel surfaces studied. higher levels were found at the higher water velocities. the biofilm attached to stainless steel was comprised of a mixed bacterial flora including Acinetobacter sp, Pseudomonas spp, Methylobacterium sp, and Corynebacterium/Arthrobacter spp. Epifluorescence microscopy provided evidence of rod-shaped bacteria and the formation of stands, possibly of extracellular material attached to stainless steel at high flow rates but not at low flow rates. Received 04 February 1998/ Accepted in revised form 12 February 1999     

Effects of Contact Time, Pressure, Percent Relative Humidity (%RH), and Material Type on Listeria Biofilm Adhesive Strength at a Cellular Level Using Atomic Force Microscopy (AFM)

Whereas the transfer of Listeria from surfaces to foods and vice versa has been well documented, little is known about the mechanism of bacterial transfer. The objective of this work is to gain a better understanding of the forces involved in listerial biofilms adhesion using atomic force microscopy (AFM). L. monocytogenes Scott A was grown as biofilms on stainless steel surfaces by inoculating stainless steel coupons with Listeria and incubating the coupons for 48 h at 32 °C with a diluted 1:20 tryptic soy broth. After growth, biofilms were equilibrated over saturated salt solutions at a constant relative humidity (%RH) before measurement of adhesion forces using AFM. The effects of contact time, loading force, and biofilm relative humidity (%RH) suggested that neither contact time, loading force nor biofilm %RH had a significant effect on biofilm adhesiveness at a cellular level (P > 0.05). In a second set of experiments, the influence of material type on biofilm adhesiveness was evaluated using two different colloidal probes (SiO₂ and polyethylene). Results showed that the maximum pull-off force and retraction work needed to retract the cantilever for glass (−85.42 nN and 1.610⁻¹⁵ J, respectively) were significantly lower than those of polyethylene (−113.38 nN and 2.7 × 10^–15 J, respectively; P < 0.001). The results of this study suggest that Listeria biofilms adhere more strongly to hydrophobic surfaces than hydrophilic surfaces when measured at a cellular level. These results provide important insights that could lead to new ways to remediate and avoid listerial biofilm formation in the food industry.     

Effects of marine paints on microbial biofilm development on three materials

The development of biofilms of Pseudomonas aeruginosa PAO-1 was studied using modified Robbins devices. Biofilm development was measured using viable counts, acridine orange direct counts (AODC), and a colorimetric method for exopolysaccharide (EPS). Biofilms reached their maximum population 24–72 h after inoculation on coupons with no paint or on coupons coated with marine paint VC-18 without additives. Biofilms on stainless steel contained higher numbers of total cells and of viable cells than biofilms on fiberglass or aluminum. Coating the surfaces with marine paint VC-18 resulted in decreased numbers of cells on stainless steel but had little effect on numbers of cells on fiberglass or aluminum. Addition to the paint of Cu or tributyltin (TBT), the active components in two types of antifouling paints, inhibited the initial development of biofilms. However, by 72–96 h, most biofilms contained the same number of cells as surfaces without additives as shown by both viable counts and AODC. Biofilms that formed on surfaces coated with Cu- or TBT-containing paint did not synthesize more EPS, suggesting that P. aeruginosa PAO-1 does not respond to these compounds by synthesizing more EPS, which could bind the metal and protect the cells. Rather, these biofilms may contain Cu- or TBT-resistant cells. TBT-resistant cells made up 1–10% of the viable counts in biofilms on uncoated stainless steel, but in biofilms on stainless steel coated with marine paint containing TBT, TBT-resistant cells made up as much as 50% of the population. For non-coated stainless steel surfaces, Cu-resistant cells initially made up the majority of the population, but after 48 h they made up less than 1% of the population. On Cu-coated stainless steel, Cu-resistant cells predominated through 48 h, but after 48 h they comprised less than 10% of the population. These results suggest that the growth of TBT-resistant and Cu-resistant cells contributes to biofilms of P. aeruginosa PAO-1 at early stages of development but not at later stages. Received 16 December 1997/ Accepted in revised form 9 March 1998     

Quantification of water and biomass in small colony variant PAO1 biofilms by confocal Raman microspectroscopy

The Raman spectra, water content, and biomass density of wild-type (WT) Pseudomonas aeruginosa PAO1, small colony variant (SCV) PAO1, and Pseudoalteromonas sp. NCIMB 2021 biofilms were compared in order to determine their variation with strain and species. Living, fully submerged biofilms were analyzed in situ by confocal Raman microspectroscopy for up to 2 weeks. Water to biomass ratios (W/BRs), which are the ratios of the O–H stretching vibration of water at 3,450 cm⁻¹ to the C–H stretching band characteristic of biomass at 2,950 cm⁻¹, were used to estimate the biomass density and cell density by comparison with W/BRs of protein solutions and bacterial suspensions, respectively, on calibration curves. The hydration within SCV biofilm colonies was extremely heterogeneous whereas W/BRs were generally constant in young WT biofilm colonies. The mean biomass in biofilm colonies of WT or colony cores of SCV was typically equivalent to 16% to 27% protein (w/v), but was 10% or less for NCIMB 2021. The corresponding cell densities were 7.5 to >10 × 10¹⁰ cfu mL⁻¹ for SCV, while the maximum cell density for NCIMB biofilms was 2.8 × 10¹⁰ cfu mL⁻¹.     

Structure and on-site formation of biofilms in paper machine water flow

Paper machine biofilms formed in situ on stainless steel surfaces were studied. A robust flow cell was fitted to side stream (1.8 m s⁻¹) of the spray water circuit of a paper machine. This on-site tool allowed for assessing the efficacy of antifoulants and the adequacy of steel polishing under mill conditions. A rapid fluorescence-based assay was developed to quantify the biomass of shallow biofilms on machine steel. The fluorescence matched the ATP content measured for the same biofilms. Electrolytic polishing reduced the tendency of biofouling of 500 grit surface steel. Biofilm grew under machine conditions as clusters on the steels, showing uniformly coccoid, filaments or short rods; only one cell type in each cluster. The biofilm clusters excluded latex beads of 0.02 μm with hydrophilic or with hydrophobic surfaces from penetrating more than three to four layers of cells. Under the high hydraulic flow at the machine (1.8 m s⁻¹), the biofilm grew in 7 days 6–10 μm thick. The high flow rate guided the shape of the biofilm clusters emerging after the primary attachment of cells. Adhered individual bacteria were the platform on steel to which solids such as paper machine fines then accumulated. Journal of Industrial Microbiology & Biotechnology (2002) 28, 268–279 DOI: 10.1038/sj/jim/7000242 Received 04 October 2001/ Accepted in revised form 14 January 2002     

Inhibiting sulfate-reducing bacteria in biofilms by expressing the antimicrobial peptides indolicidin and bactenecin

To identify novel, less-toxic compounds capable of inhibiting sulfate-reducing bacteria (SRB), Desulfovibrio vulgaris and Desulfovibrio gigas in suspension cultures were exposed to several antimicrobial peptides. The bacterial peptide antimicrobials gramicidin S, gramicidin D, and polymyxin B as well as the cationic peptides indolicidin and bactenecin from bovine neutrophils decreased the viability of both SRB by 90% after a 1-h exposure at concentrations of 25–100 μg ml⁻¹. To reduce corrosion by inhibiting SRB in biofilms, the genes for indolicidin and bactenecin were expressed in Bacillus subtilisBE1500 and B. subtilis WB600 under the control of the constitutive alkaline protease (apr) promoter, and the antimicrobials were secreted into the culture medium using the apr signal sequence. Bactenecin was also synthesized and expressed as a fusion to the pro-region of barnase from Bacillus amyloliquefaciens. Concentrated culture supernatants of B. subtilis BE1500 expressing bactenecin at 3 μg ml⁻¹ decreased the viability of Escherichia coli BK6 by 90% and the reference SRB D. vulgaris by 83% in suspension cultures. B. subtilis BE1500 and B. subtilis WB600 expressing bactenecin in biofilms also inhibited the SRB-induced corrosion of 304 stainless steel six to 12-fold in continuous reactors as evidenced by the lack of change in the impedance spectra (resistance polarization) upon addition of SRB and by the reduction in hydrogen sulfide and iron sulfide in batch fermentations with mild steel. A 36-fold decrease in the population of D. vulgaris in a B. subtilis BE1500 biofilm expressing bactenecin was also observed. This is the first report of an antimicrobial produced in a biofilm for in vivo applications and represents the first application of a beneficial, genetically-engineered biofilm for combating corrosion. Received 27 October 1998/ Accepted in revised form 21 February 1999     

Temperature dependence of habituation of the initial responses to cold-water immersion

The initial responses to cold-water immersion, evoked by stimulation of peripheral cold receptors, include tachycardia, a reflex inspiratory gasp and uncontrollable hyperventilation. When immersed naked, the maximum responses are initiated in water at 10°C, with smaller responses being observed following immersion in water at 15°C. Habituation of the initial responses can be achieved following repeated immersions, but the specificity of this response with regard to water temperature is not known. Thirteen healthy male volunteers were divided into a control (C) group (n = 5) and a habituation (H) group (n = 8). Each subject undertook two 3-min head-out immersions in water at 10°C wearing swimming trunks. These immersions took place at a corresponding time of day with 4 days separating the two immersions. In the intervening period the C group were not exposed to cold water, while the H group undertook another six, 3-min, head-out immersions in water at 15°C. Respiratory rate (f _R), inspiratory minute volume (V˙ _I) and heart rate (f _H) were measured continuously throughout each immersion. Following repeated immersions in water at 15°C, the f _R, V˙ _I and f _H responses of the H group over the first 30 s of immersion were reduced (P < 0.01) from 33.3 breaths · min⁻¹, 50.5 l · min⁻¹ and 114 beats · min⁻¹ respectively, to 19.8 breaths · min⁻¹, 26.4 l · min⁻¹ and 98 beats · min⁻¹, respectively. In water at 10°C these responses were reduced (P < 0.01) from 47.3 breaths · min⁻¹, 67.6 l · min⁻¹ and 128 beats · min⁻¹ to 24.0 breaths · min⁻¹, 29.5 l · min⁻¹ and 109 beats · min⁻¹, respectively over a corresponding period of immersion. Similar reductions were observed during the last 2.5 min of immersions. The initial responses of the C group were unchanged. It is concluded that habituation of the cold shock response can be achieved by immersion in warmer water than that for which protection is required. This suggests that repeated submaximal stimulation of the cutaneous cold receptors is sufficient to attenuate the responses to more maximal stimulation. Accepted: 6 February 1998     

Destruction of Deinococcus geothermalis biofilm by photocatalytic ALD and sol-gel TiO2 surfaces

The aim of the present work was to explore possibilities of photocatalytic TiO₂ coating for reducing biofilms on non-living surfaces. The model organism, Deinococcus geothermalis, known to initiate growth of durable, colored biofilms on machine surfaces in the paper industry, was allowed to form biofilms on stainless steel, glass and TiO₂ film coated glass or titanium. Field emission electron microscopy revealed that the cells in the biofilm formed at 45°C under vigorous shaking were connected to the surface by means of numerous adhesion threads of 0.1--0.3 μm in length. Adjacent cells were connected to one another by threads of 0.5--1 μm in length. An ultrastructural analysis gave no indication for the involvement of amorphous extracellular materials (e.g., slime) in the biofilm. When biofilms on photocatalytic TiO₂ surfaces, submerged in water, were exposed to 20 W h m⁻² of 360 nm light, both kinds of adhesion threads were completely destroyed and the D. geothermalis cells were extensively removed (from >10⁷ down to below 10⁶ cells cm⁻²). TiO₂ films prepared by the sol-gel technique were slightly more effective than those prepared by the ALD technique. Doping of the TiO₂ with sulfur did not enhance its biofilm-destroying capacity. The results show that photocatalytic TiO₂ surfaces have potential as a self-cleaning technology for warm water using industries.     

Bioreduction of Cu(II) by Cell-Free Copper Reductase from a Copper Resistant <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NA

Environmental copper contamination is a serious human health problem. Copper reductase is produced by microorganisms to facilitate copper uptake by ATPases into the cells increasing copper biosorption. This study assessed the reduction of Cu(II) by cell-free extracts of a highly copper-resistant bacterium, Pseudomonas sp. strain NA, isolated from vineyard soil contaminated with copper. Both intact cells and cell-free extract of Pseudomonas sp. strain NA displayed substantial reduction of Cu(II). Intact cells reduced more then 80 mg L⁻¹ of Cu(II) from medium amended with 200 mg L⁻¹ of copper after 24 h of incubation. Cell-free extract of the isolate reduced more than 65% of the Cu(II) at initial copper concentration of 200 mg L⁻¹ after 24 h. Soluble protein production was high at 72 h of incubation at 100 mg L⁻¹ of copper, with more then 60 μg L⁻¹ of total soluble protein in cell-free extract recorded. Cu(II) reduction by isolate NA was increased when copper concentration increased for both intact cells and cell-free extract. Results indicate that Pseudomonas sp. strain NA produces copper reductase enzyme as the key mechanism of copper biotransformation.     

Cardiorespiratory responses to underwater treadmill walking in healthy females

This study compared the cardiorespiratory responses of eight healthy women (mean age 30.25 years) to submaximal exercise on land (LTm) and water treadmills (WTm) in chest-deep water (Aquaciser). In addition, the effects of two different water temperatures were examined (28 and 36°C). Each exercise test consisted of three consecutive 5-min bouts at 3.5, 4.5 and 5.5 km · h⁻¹. Oxygen consumption (V˙O₂) and heart rate (HR), measured using open-circuit spirometry and telemetry, respectively, increased linearly with increasing speed both in water and on land. At 3.5 km · h⁻¹ V˙O₂ was similar across procedures [χ = 0.6 (0.05) l · min⁻¹]. At 4.5 and 5.5 km · h⁻¹ V˙O₂ was significantly higher in water than on land, but there was no temperature effect (WTm: 0.9 and 1.4, respectively; LTm: 0.8 and 0.9 l · min⁻¹, respectively). HR was significantly higher in WTm at 36°C compared to WTm at 28°C at all speeds, and compared to LTm at 4.5 and 5.5 km · h⁻¹ (P ≤ 0.003). The HR-V˙O₂ relationship showed that at a V˙O₂ of 0.9 l · min⁻¹, HR was higher in water at 36°C (115 beats · min⁻¹) than either on land (100 beats · min⁻¹) or in water at 28°C (99 beats · min⁻¹). The Borg scale of perceived exertion showed that walking in water at 4.5 and 5.5 km · h⁻¹ was significantly harder than on land (WTm: 11.4 and 14, respectively; LTm: 9.9 and 11, respectively; P ≤ 0.001). These cardiorespiratory changes occurred despite a slower cadence in water (the mean difference at all speeds was 27 steps/min). Thus, walking in chest-deep water yields higher energy costs than walking at similar speeds on land. This data has implications for therapists working in hydrotherapy pools. Accepted: 3 September 1997     

Stability of diatom composition in a variable lake environment: Lake Chascomús, Argentina

 Water and surficial sediment samples of Lake Chascomús and its tributaries were analyzed in order to relate changes in diatom community structure to chemical variables. Over the course of 13 months of sampling, the lake exhibited major changes in water level (1.15–1.98 m average depth), total dissolved solids (821–1972 mg l⁻¹), silica (0.098–8.22 mg l⁻¹), and total algal biomass (21.4–145.9 μg Chl a l⁻¹). However, despite these large fluctuations, the diatom species composition was relatively stable. The dominant species in the water column was always Synedra berolinensis (68.9%–90.1% total frustules), with Fragilaria construens and F. brevistriata as subdominants. In the sediments the latter two species dominated the frustule counts. These results indicate an unusual floristic stability of this eutrophic ecosystem, with persistent dominance by broadly tolerant, generalist species. Received: April 24, 2001 / Accepted: January 23, 2002     

Water and sodium balances and metabolic physiology of house mice (Mus domesticus) and short-tailed mice (Leggadina lakedownensis) under laboratory conditions

A laboratory study investigated the metabolic physiology, and response to variable periods of water and sodium supply, of two arid-zone rodents, the house mouse (Mus domesticus) and the Lakeland Downs short-tailed mouse (Leggadina lakedownensis) under controlled conditions. Fractional water fluxes for M. domesticus (24 ± 0.8%) were significantly higher than those of L. lakedownensis (17 ± 0.7%) when provided with food ad libitum. In addition, the amount of water produced by M. domesticus and by L. lakedownensis from metabolic processes (1.3 ± 0.4 ml · day⁻¹ and 1.2 ± 0.4 ml · day⁻¹, respectively) was insufficient to provide them with their minimum water requirement (1.4 ± 0.2 ml · day⁻¹ and 2.0 ± 0.3 ml · day⁻¹, respectively). For both species of rodent, evaporative water loss was lowest at 25 °C, but remained significantly higher in M. domesticus (1.1 ± 0.1 mg H₂O · g^−0.122 · h⁻¹) than in L. lakedownensis (0.6 ± 0.1 mg H₂O · g^−0.122 · h⁻¹). When deprived of drinking water, mice of both species initially lost body mass, but regained it within 18 days following an increase in the amount of seed consumed. Both species were capable of drinking water of variable saline concentrations up to 1 mol · l⁻¹, and compensated for the increased sodium in the water by excreting more urine to remove the sodium. Basal metabolic rate was significantly higher in M. domesticus (3.3 ± 0.2 mg O₂ · g^−0.75 · h⁻¹) than in L. lakedownensis (2.5 ± 0.1 mg O₂ · g^−0.75 · h⁻¹). The study provides good evidence that water flux differences between M. domesticus and L. lakedownensis in the field are due to a requirement for more water in M. domesticus to meet their physiological and metabolic demands. Sodium fluxes were lower than those observed in free-ranging mice, whose relatively high sodium fluxes may reflect sodium associated with available food. Accepted: 16 August 1999     

Effect of flow rate on heavy metal accumulation by rotating biological contactor (RBC) biofilms

Immobilized biofilms are effective in heavy metal removal. The current studies investigated the use of rotating biological contactor (RBC) biofilms in treatment of a wastewater containing cadmium, copper and zinc, each at a concentration of 100 mg L⁻¹. In particular, the influence of hydraulic retention time (HRT) on metal accumulation was studied. Longer HRTs (>12 h) were associated with greater metal removal than short HRTs, particularly with regard to cadmium and zinc. The system was also shown to operate successfully over an extended period of time, at an HRT of 24 h, with removal efficiencies of approximately 34%, 85% and 57% for Cd²⁺, Cu²⁺ and Zn²⁺ respectively after 5–8 weeks contact. Journal of Industrial Microbiology & Biotechnology (2000) 24, 244–250. Received 28 July 1999/ Accepted in revised form 21 December 1999     

Physiology and kinetics of trimethylamine conversion by two methylotrophic strains in continuous cultivation systems

The change of dilution rate (D) on both Methylophilus methylotrophus NCIMB11348 and Methylobacterium sp. RXM CCMI908 growing in trimethylamine (TMA) chemostat cultures was studied in order to assess their ability to remove odours in fish processing plants. M. methylotrophus NCIMB11348 was grown at dilution rates of 0.012–0.084 h⁻¹ and the biomass level slightly increased up to values of D around 0.07 h⁻¹. The maximum cell production rate was obtained at 0.07 h⁻¹ corresponding to a maximum conversion of carbon into cell mass (35%). The highest rate of TMA consumption was 3.04 mM h⁻¹ occurring at D=0.076 h⁻¹. Methylobacterium sp. RXM CCMI908 was grown under similar conditions. The biomass increased in a more steep manner up to values of D around 0.06 h⁻¹. The maximum cell production rate (0.058 g l⁻¹h⁻¹) was obtained in the region close to 0.06 h⁻¹ where a maximum conversion of the carbon into cell mass (40%) was observed. The maximum TMA consumption was 2.33 mM h⁻¹ at D=0.075 h⁻¹. The flux of carbon from TMA towards cell synthesis and carbon dioxide in both strains indicates that the cell is not excreting products but directing most of the carbon source to growth. Carbon recovery levels of approximately 100% show that the cultures are carbon-limited. Values for theoretical maximum yields and maintenance coefficients are presented along with a kinetic assessment based on the determination of the substrate saturation constant and maximum growth rate for each organism. Received: 25 February 1999 / Received revision: 14 May 1999 / Accepted: 17 May 1999     

Water influx and efflux in free-flying pigeons

We used tritium-labeled water to measure total body water, water influx (which approximated oxidative water production) and water efflux in free-flying tippler pigeons (Columba livia) during flights that lasted on average 4.2 h. At experimental air temperatures ranging from 18 to 27 °C, mean water efflux by evaporation and excretion [6.3 ± 1.3 (SD) ml · h⁻¹, n = 14] exceeded water influx from oxidative water and inspired air (1.4 ± 0.7 ml · h⁻¹, n = 14), and the birds dehydrated at 4.9 ± 0.9 ml · h⁻¹. This was not significantly different from gravimetrically measured mass loss of 6.2 ± 2.1 g · h⁻¹ (t = 1.902, n = 14, P>0.05). This flight-induced dehydration resulted in an increase in plasma osmolality of 4.3 ± 3.0 mosmol · kg⁻¹ · h⁻¹ during flights of 3–4 h. At 27 °C, the increase in plasma osmolality above pre-flight levels (ΔP _osm = 7.6±4.29 mosmol · kg⁻¹ · h⁻¹, n = 6) was significantly higher than that at 18 °C (ΔP _osm = 0.83±2.23 mosmol · kg⁻¹ · h⁻¹, (t = 3.43, n = 6, P < 0.05). Post-flight haematocrit values were on average 1.1% lower than pre-flight levels, suggesting plasma expansion. Water efflux values during free flight were within 9% of those in the one published field study (Gessaman et al. 1991), and within the range of values for net water loss determined from mass balance during wind tunnel experiments (Biesel and Nachtigall 1987). Our net water loss rates were substantially higher than those estimated by a simulation model (Carmi et al. 1992) suggesting some re-evaluation of the model assumptions is required. Accepted: 8 April 1997     

Evaluation of antioxidant properties and total phenolic contents of some strains of microalgae

Antioxidant activities of both cells and extracellular substances were evaluated in 12 soil-isolated strains of microalgae according to FRAP and DPPH-HPLC assays. Their total phenolic contents were also determined by Folin–Ciocalteu method. Extractions were performed with hexane, ethyl acetate, and water. The results of FRAP assay showed that algal cells contained considerable amounts of antioxidants from 0.56 ± 0.06 to 31.06 ± 4.00 μmol Trolox g⁻¹ for Microchaete tenera hexane extract and Chlorella vulgaris water extract, respectively. In water fractions of extracellular substances, the antioxidants were from 1.30 ± 0.15 μmol Trolox g⁻¹ for Fischerella musicola to 73.20 ± 0.16 μmol Trolox g⁻¹ for Fischerella ambigua. Also, DPPH-HPLC assay represented high antioxidant potential of water fractions. The measured radical-scavenging activities of the studied microalgae were at least 0.15 ± 0.02 in Nostoc ellipsosporum cell mass to a maximum of 109.02 ± 8.25 in C. vulgaris extracellular substance. The amount of total phenolic contents varied in different strains of microalgae and ranged from zero in hexane extract to 19.15 ± 0.04 mg GAE g⁻¹ in C. vulgaris extracellular water fraction. Significant correlation coefficients between two measured parameters indicated that phenolic compounds were a major contributor to the microalgal antioxidant capacities.     

Studies on water transport through the sweet cherry fruit surface: characterizing conductance of the cuticular membrane using pericarp segments

Water conductance of the cuticular membrane (CM) of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated by monitoring water loss from segments of the outer pericarp excised from the cheek of the fruit. Segments consisted of epidermis, hypodermis and several cell layers of the mesocarp. Segments were mounted in stainless-steel diffusion cells with the mesocarp surface in contact with water, while the outer cuticular surface was exposed to dry silica (22 ± 1 °C). Conductance was calculated by dividing the amount of water transpired per unit area and time by the difference in water vapour concentration across the segment. Conductance values had a log normal distribution with a median of 1.15 × 10⁻⁴ m s⁻¹ (n=357). Transpiration increased linearly with time. Conductance remained constant and was not affected by metabolic inhibitors (1 mM NaN₃ or 0.1 mM carbonylcyanide m-chlorophenylhydrazone) or thickness of segments (range 0.8–2.8 mm). Storing fruit (up to 42 d, 1 °C) used as a source of segments had no consistent effect on conductance. Conductance of the CM increased from cheek (1.16 ± 0.10 × 10⁻⁴ m s⁻¹) to ventral suture (1.32 ± 0.07 × 10⁻⁴ m s⁻¹) and to stylar end (2.53 ± 0.17 × 10⁻⁴ m s⁻¹). There was a positive relationship (r²=0.066**; n=108) between conductance and stomatal density. From this relationship the cuticular conductance of a hypothetical astomatous CM was estimated to be 0.97 ± 0.09 × 10⁻⁴ m s⁻¹. Removal of epicuticular wax by stripping with cellulose acetate or extracting epicuticular plus cuticular wax by dipping in CHCl₃/methanol increased conductance 3.6- and 48.6-fold, respectively. Water fluxes increased with increasing temperature (range 10–39 °C) and energies of activation, calculated for the temperature range from 10 to 30 °C, were 64.8 ± 5.8 and 22.2 ± 5.0 kJ mol⁻¹ for flux and vapour-concentration-based conductance, respectively. Received: 23 March 2000 / Accepted: 28 July 2000     

Effects of Acetic Acid on the Regrowth of Heterotrophic Bacteria in the Drinking Water Distribution System

Summary Three laboratory-scale water pipe systems were set up to study the effects of adding two levels of acetic acid (10 and 50 μg acetate eq-C l⁻¹) on the bacterial regrowth in water pipes. The results of the water pipe test showed that nearly all carbon in the acetic acid could be readily utilized by bacteria and resulted in an increase in biomass concentration. The maximum heterotrophic plate counts in biofilm were equal to 3.5 × 10⁴, 8.9 × 10⁵ and 2.9 × 10⁷ c.f.u. cm⁻² while the maximum heterotrophic plate counts of free bacteria were equal to 1.2 × 10³, 5.0 × 10³ and 6.8 × 10⁴ c.f.u. ml⁻¹ for the blank and with addition of 10 and 50 μg acetate eq-C l⁻¹. These results showed that addition of acetic acid to drinking water has a positive effect on the assimilable organic carbon content of drinking water and bacterial regrowth in the distribution system. This effect is enhanced with addition of high-level acetic acid. Batch tests were also conducted using water samples collected from a Taiwanese drinking water distribution system. The bacterial regrowth potentials of the blank were equal to 4.3 × 10³, 1.5 × 10⁴, 4.9 × 10⁴ and 7.5 × 10⁴ c.f.u. ml⁻¹ for water samples collected from treatment plant effluent, commercial area, mixed area, and residential area, respectively. These results showed that the biological stability of drinking water is the highest in treatment plant effluent, followed by distributed water of the commercial area, distributed water of the mixed area, and then the distributed water of residential area.     

Asian small-clawed otters (Amblonyx cinerea): resting and swimming metabolic rates

Open-flow oxygen and carbon dioxide respirometry was used in Neumünster Zoo (Germany) to examine the energy requirements of six Asian small-clawed otters (Amblonyx cinerea) at rest and swimming voluntarily under water. Our aim was to compare their energy requirements with those of other warm-blooded species to elucidate scale effects and to test whether the least aquatic of the three otter species differs markedly from these and its larger relatives. While at rest on land (16 °C, n = 26), otters (n = 6, mean body mass 3.1 ± 0.4 kg) had a respiratory quotient of 0.77 and a resting metabolic rate of 5.0 ± 0.8 Wkg⁻¹(SD). This increased to 9.1 ± 0.8 Wkg⁻¹ during rest in water (11–15 °C, n = 4) and to 17.6 ± 1.4 Wkg⁻¹ during foraging and feeding activities in a channel (12 °C, n = 5). While swimming under water (n = 620 measurements) in an 11-m long channel, otters preferred a speed range between 0.7 ms⁻¹ and 1.2 ms⁻¹. Transport costs were minimal at 1 ms⁻¹ and amounted to 1.47 ± 0.24 JN⁻¹ m⁻¹ (n = 213). Metabolic rates of small-clawed otters in air were similar to those of larger otter species, and about double those of terrestrial mammals of comparable size. In water, metabolic rates during rest and swimming were larger than those extrapolated from larger otter species and submerged swimming homeotherms. This is attributed to high thermoregulatory costs, and high body drag at low Reynolds numbers. Accepted: 21 December 1998