Complete degradation of tetrachloroethene in coupled anoxic and oxic chemostats

Anaerobic tetrachloroethene(C₂Cl₄)-dechlorinating bacteria were enriched in slurries from chloroethene-contaminated soil. With methanol as electron donor, C₂Cl₄ and trichloroethene (C₂HCl₃) were reductively dechlorinated to cis-1,2-dichloroethene (cis-C₂H₂Cl₂), whereas, with l-lactate or formate, complete dechlorination of C₂Cl₄ via C₂HCl₃, cis-C₂H₂Cl₂ and chloroethene (C₂H₃Cl) to ethene was obtained. In oxic soil slurries with methane as a substrate, complete co-metabolic degradation of cis-C₂H₂Cl₂ was obtained, whereas C₂HCl₃ was partially degraded. With toluene or phenol both of the above were readily co-metabolized. Complete degradation of C₂Cl₄ was obtained in sequentially coupled anoxic and oxic chemostats, which were inoculated with the slurry enrichments. Apparent steady states were obtained at various dilution rates (0.02–0.4 h⁻¹) and influent C₂Cl₄-concentrations (100–1000 μM). In anoxic chemostats with a mixture␣of␣formate and glucose as the carbon and electron source, C₂Cl₄ was transformed at high rates (above␣140 μmol l⁻¹ h⁻¹, corresponding to 145 nmol Cl⁻ min⁻¹ mg protein⁻¹) into cis-C₂H₂Cl₂ and C₂H₃Cl. Reductive dechlorination was not affected by addition of 5 mM sulphate, but strongly inhibited after addition of 5 mM nitrate. Our results (high specific dechlorination rates and loss of dechlorination capacity in the absence of C₂Cl₄) suggest that C₂Cl₄-dechlorination in the anoxic chemostat was catalysed by specialized dechlorinating bacteria. The partially dechlorinated intermediates, cis-C₂H₂Cl₂ and C₂H₃Cl, were further degraded by aerobic phenol-metabolizing bacteria. The maximum capacity for chloroethene (the sum of tri-, di- and monochloro derivatives removed) degradation in the oxic chemostat was 95 μmol l⁻¹ h⁻¹ (20 nmol min⁻¹ mg protein⁻¹), and that of the combined anoxic → oxic reactor system was 43.4 μmol l⁻¹ h⁻¹. This is significantly higher than reported thus far. Received: 17 April 1997 / Received revision: 6 June 1997 / Accepted: 7 June 1997     

Properties of tetrachloroethene and trichloroethene dehalogenase of Dehalospirillum multivorans

Some properties of tetrachloroethene and trichloroethene dehalogenase of the recently isolated, tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, were studied with extracts of cells grown on pyruvate plus fumarate. The dehalogenase catalyzed the oxidation of reduced methyl viologen with tetrachloroethene (PCE) or trichloroethene (TCE) as electron acceptor. All other artificial or physiological electron donors tested were ineffective. The PCE and TCE dehalogenase activity was insensitive towards oxygen in crude extracts. When extracts were incubated under anoxic conditions in the presence of titanium citrate as reducing agent, the dehalogenase was rapidly inactivated by propyl iodide (50 M). Inactivation did not occur in the absence of titanium citrate. The activity of propyl-iodide-treated extracts was restored almost immediately by illumination. The dehalogenase was inhibited by cyanide. The inhibition profile was almost the same under oxic and anoxic conditions independent of the presence or absence of titanium citrate. In addition, N₂O, nitrite, and ethylene diamine tetra-acetate (EDTA) were inhibitors of PCE and TCE dehalogenase. Carbon monoxide and azide had no influence on the dehalogenase activity. Trans-1,2-dichloroethene or 1,1-dichloroethene, both of which are isomers of the dechlorination product cis-1,2-dichloroethene, neither inhibited nor inactivated the dehalogenase. PCE and TCE dechlorination appeared to be mediated by the same enzyme since the inhibitors tested had nearly the same effects on the PCE and TCE dehalogenating activity. The data indicated the involvement of a corrinoid and possibly of an additional transition metal in reductive PCE and TCE dechlorination.Abbreviations PCE Tetrachloroethene - TCE Trichloroethene - DCE Dichloroethene - EDTA Ethylene diamine tetra-acetate - MV Methyl viologen - BV Benzyl viologen - PI Propyl iodide, 1-iodopropane - TC Titanium(III) citrate     

Diatom composition and biomass variability in nearshore waters of Maxwell Bay, Antarctica, during the 1992/1993 austral summer

 Diatom composition and biomass were investigated in the nearshore water (<30 m in depth) of Maxwell Bay, Antarctica during the 1992/1993 austral summer. Epiphytic or epilithic diatoms such as Fragilaria striatula, Achnanthes brevipes var. angustata and Licmophora spp. dominated the water column microalgal populations. Within the bay, diatom biomass in surface water was several times higher at the nearshore (2.4–14 μg C l^-1) than at the offshore stations (>100 m) (1.2–3.2 μg C l^-1) with a dramatic decrease towards the bay mouth. Benthic forms accounted for >90% of diatom carbon in all nearshore stations, while in the offshore stations planktonic forms such as Thalassiosira antarctica predominated (50–>90%). Microscopic examination revealed that many of these diatoms have become detached from a variety of macroalgae growing in the intertidal and shallow subtidal bottoms. Epiphytic diatoms persistently dominated during a 19-day period in the water column at a fixed nearshore station, and the biomass of these diatoms fluctuated from 0.86 to 53 μg C l^-1. A positive correlation between diatom biomass and wind speed strongly suggests that wind-driven resuspension of benthic forms is the major mechanism increasing diatom biomass in the water column. Received: 28 April 1995/Accepted: 1 April 1996     

The microbial plankton of Lake Fryxell,southern Victoria Land,Antarctica during the summers of 1992 and 1994

 Samples collected from Lake Fryxell, southern Victoria Land, Antarctica in January 1992 and 1994 were analysed for the abundance of bacterioplankton and the diversity and abundance of protistan plankton. At the times of sampling, 14 ciliate species and 10 species of autotrophic flagellate were recorded. The samples contained two species of rotifer (Philodina spp.), which formed the first record of planktonic metazoans in the Dry Valley lakes of this region of Antarctica. Bacterial concentrations ranged between 1.0 and 3.8×10⁸ l^-1 in the upper oxic waters increasing to 20×10⁸ l^-1 in the anoxic waters. Heterotrophic flagellates decreased in abundance down the oxygenated water column, disappearing completely at 9 m, and ranged between 0.28 and 7.39×10⁵ l^-1 in abundance. Autotrophic flagellates were much more abundant exhibiting a number of distinct peaks down the water column (1.89–25.3×10⁸ l^-1). The ciliated protozoa were very abundant (up to 7720 l^-1) in relation to flagellate and bacterial numbers, typical of oligotrophic lakes world-wide. The distribution of the protistan plankton showed marked zonation, probably in response to the differing salinity and temperature gradients in the water column. Possible trophic interactions are discussed and comparisons with other continental Antarctic lakes made. Received: 29 November 1995/Accepted: 18 February 1996     

Tetrachloroethene and 3-chlorobenzoate dechlorination activities are co-induced inDesulfomonile tiedjei DCB-1

Desulfomonile tiedjei, a strict anaerobe capable of reductively dechlorinating 3-chlorobenzoate, also dechlorinates tetrachloroethene and trichloroethene. It is not known, however, if the aryl and aliphatic dechlorination activities are catalyzed by the same enzymatic system. Cultures induced for 3-chlorobenzoate activity dechlorinated tetrachloroethene and trichloroethene to lower chlorinated products while uninduced parallel cultures did not dechlorinate either substrate. The observed rate of PCE dechlorination in induced cultures was 22 µmol h^–1 g protein^–1, which is considerably faster than previous rates obtained with defined cultures of this organism. These results show that both dechlorination activities are co-induced and therefore, that the dechlorination mechanisms may share at least some components.Abbreviations PCE tetrachloroethene - TCE trichloroethene - cis-DCE cis-dichloroethene - trans-DCE trans-dichloroethene - 3FBz 3-fluorobenzoate - 3ClBz 3-chlorobenzoate     

Effects of saturated fatty acids on amylase release from exocrine pancreatic segments of sheep,rats, hamsters,field voles and mice

 Stimulatory effects of saturated fatty acids consisting of 4 (butyrate), 8 (octanoate), 12 (laurate) and 16 (palmitate) carbon atoms, as well as acetylcholine on pancreatic amylase release were assessed in tissue segments isolated from sheep, rats, hamsters, field voles and mice. The amount of amylase release induced by the fatty acids (1 μmol ⋅ l^-1 to 10 mml ⋅ l^-1) and by acetylcholine (10 nmol ⋅ l^-1 to 100 μmol ⋅ l^-1) increased in a concentration-dependent manner, and the maximum response in response to the fatty acids was obtained at the maximal dose used. The maximum increase in amylase release in response to butyrate or octanoate was highly and significantly (r=0.974, P<0.001) dependent on the log value of the mean body mass in the following order: sheep>rats>hamsters>field voles>mice. On the other hand, the response to laurate and palmitate was variable among animal species. Addition of atropine (1.4 μmol ⋅ l^-1) to the medium did not reduce the responses to octanoate stimulation, but significantly reduced acetylcholineinduced responses, implying that the effects of the fatty acids were not mediated through activation of muscarinic acetylcholine receptors. Reduction of calcium ion concentration in the medium significantly inhibited the responses induced by the fatty acids and acetylcholine, suggesting that amylase release depends on extracellular calcium ions. Accepted: 14 May 1996     

A highly purified enrichment culture couples the reductive dechlorination of tetrachloroethene to growth.

A microscopically pure enrichment culture of a gram-negative anaerobic bacterium, in the present article referred to as PER-K23, was isolated from an anaerobic packed-bed column in which tetrachloroethene (PCE) was reductively transformed to ethane via trichloroethene (TCE), cis-1,2-dichloroethene (cis-1,2-DCE), chloroethene, and ethene. PER-K23 catalyzes the dechlorination of PCE via TCE to cis-1,2-DCE and couples this reductive dechlorination to growth. H2 and formate were the only electron donors that supported growth with PCE or TCE as an electron acceptor. The culture did not grow in the absence of PCE or TCE. Neither O2, NO3-, NO2-, SO4(2-), SO3(2-), S2O3(2-), S, nor CO2 could replace PCE or TCE as an electron acceptor with H2 as an electron donor. Also, organic electron acceptors such as acetoin, acetol, dimethyl sulfoxide, fumarate, and trimethylamine N-oxide and chlorinated ethanes, DCEs, and chloroethene were not utilized. PER-K23 was not able to grow fermentatively on any of the organic compounds tested. Transferring the culture to a rich medium revealed that a contaminant was still present. Dechlorination was optimal between pH 6.8 and 7.6 and a temperature of 25 to 35 degrees C. H2 consumption was paralleled by chloride production, PCE degradation, cis-1,2-DCE formation, and growth of PER-K23. Electron balances showed that all electrons derived from H2 or formate consumption were recovered in dechlorination products and biomass. Exponential growth could be achieved only in gently shaken cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene-Respiring Dehalococcoides Isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 μmol liter⁻¹ day⁻¹, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ± 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.     

Scale-up issues for in situ anaerobic tetrachloroethene bioremediation

For the full scale implementation of in situ anaerobic bioremediation of tetrachloroethene (PCE) in groundwater, the following issues must be addressed: which organic substrates at which concentration would be most effective in promoting dechlorination and are economical; how far the substrate, electron acceptor, and nutrients can be transported in the aquifer; and the placement of delivery and recovery wells for distributing these amendments. In a microcosm study, almost all of the tested inexpensive substrates supported reductive dechlorination of PCE through vinyl chloride (VC) under methanogenic conditions. A minimum of about 60 mg L⁻¹ of organic carbon was needed to dechlorinate 23 μM PCE with a single feeding. In a second microcosm study dechlorination stopped at 1,2-dichloroethene (DCE) in microcosms fed higher concentrations of several substrates. At the highest concentrations the substrates inhibited DCE production. Three field tracer tests were conducted to evaluate methods to distribute the amendments across the aquifer. The natural groundwater gradient is not sufficient to distribute substrate evenly. Groundwater injection at 60 times the natural flux rate increased the distribution of substrate. A mixing strategy of cross-gradient injection further increased the distribution of the substrate. Ammonia-nitrogen, sulfate, and phosphate were retarded relative to the substrate and inorganic tracer. Received 30 October 1995/ Accepted in revised form 07 June 1996     

Picophytoplankton biomass, community structure and productivity in the Great Astrolabe Lagoon, Fiji

 Phytoplankton biomass, community structure and productivity of the Great Astrolabe lagoon and surrounding ocean were studied using measurements of chlorophyll concentration and carbon uptake. The contribution of picophytoplankton to biomass, productivity and community structure was estimated by size fractionation, ¹⁴C-incubation and flow cytometry analysis. Picoplankton red fluorescence was demonstrated to be a proxy for chlorophyll <3 μm. Consequently, the percentage contribution to chl a<3 μm from each picoplankton group could be calculated using regression estimated values of ψ _i (fg chl a per unit of red fluorescence). In the lagoon, average chlorophyll concentration was 0.8 mg m^-3 with 45% of phytoplankton <3 μm. Primary production reached 1.3 g C m^-2 day^-1 with 53% due to phytoplankton <3 μm. Synechococcus was the most abundant group at all stations, followed by Prochlorococcus and picoeukaryotes. At all stations, Prochlorococcus represented less than 4% of the chl a <3 μm, Synechococcus between 85 and 95%, and Picoeukaryotes between 5 and 10%. In the upper 40 m of surrounding oceanic waters, phytoplankton biomass was dominated by the >3 μm size fraction. In deeper water, the <1 μm size fraction dominated. Prochlorococcus was the most abundant picoplankton group and their contributions to the chlorophyll a<3 μm were close to that of the picoeukaryotes (50% each). Accepted: 27 May 1999     

Production of poly(3-hydroxyalkanoates) by Pseudomonas putida KT2442 in continuous cultures

 The synthesis of poly(3-hydroxyalkanoates) (PHA) by Pseudomonas putida KT2442 growing on long-chain fatty acids was studied in continuous cultures. The effects of the growth rate on the biomass and polymer concentration were determined and it was found that the PHA concentrations decreased with increasing growth rates. The highest volumetric productivity was 0.13 g PHA l^-1 h^-1 at a specific growth rate (μ) of 0.1 h^-1. The molecular mass of the polymer remained constant at all growth rates but changes in the monomeric composition of the PHA synthesized were observed. Variation of the carbon to nitrogen (C/N) ratio of the substrate feed at μ=0.1 h^-1 revealed optimal PHA formation at C/N=20 mol/mol. In order to optimize PHA production P. putida KT2442 was cultivated to high cell densities in oxygen-limited continuous cultures. In this way a maximum biomass concentration of 30 g/l containing approximately 23% PHA was achieved. This corresponds to a volumetric productivity of 0.69 g  l^-1 h^-1. Received: 14 December 1995 / Received revision: 18 April 1996 / Accepted: 22 April 1996     

Anaerobic dechlorination of trichloroethene,tetrachloroethene and 1,2-dichloroethane by an acetogenic mixed culture in a fixed-bed reactor

An anaerobic enrichment culture with glucose as the sole source of carbon and energy plus trichloroethene (TCE) as a potential electron acceptor was inoculated with material from a full size anaerobic charcoal reactor that biologically eliminated dichloromethane from contaminated groundwater (Stromeyer et al. 1991). In subcultures of this enrichment complete sequential transformation of 10 µM TCE viacis-dichloroethene and chloroethene to ethene was reproducibly observed. Maintenance of this activity on subcultivation required the presence of TCE in the medium. The enrichment culture was used to inoculate an anaerobic fixed-bed reactor containing sintered glass Raschig elements as support material. The reactor had a total volume of 1780 ml and was operated at 20 °C in an up-flow mode with a flow rate of 50 ml/h. It was fed continuously with 2 mM glucose and 55 µM TCE. Glucose was converted to acetate as the major product and to a minor amount of methane; TCE was quantitatively dehalogenated to ethene. When, in addition to TCE, tetrachloroethene or 1,2-dichloroethane were added to the system, these compounds were also dehalogenated to ethene. In contrast, 1,1,1-trichloroethane was not dehalogenated, but at 40 µM severely inhibited acetogenesis and methanogenesis. When the concentration of TCE in the feed was raised to 220 µM, chloroethene transiently accumulated, but after an adaptation period ethene was again the only volatile product detected in the effluent. The volumetric degradation rate at this stage amounted to 6.2 µmol/l/h. Since complete transformation of TCE occurred in the first sixth of the reactor volume, the degradation capacity of the system is estimated to exceed this value by factor of about ten.Abbreviations CA chloroethane - 1,1-DCA 1,1-dichloroethane - 1,2-DCA 1,2-dichloroethane - 1,1-DCE 1,1-dichloroethene - c-DCE cis-1,2-dichloroethene - t-DCE trans-1,2-dichloroethene - PCE tetrachloroethene, perchloroethene - 1,1,1-TCA 1,1,1-trichloroethane - TCE trichloroethene - VC chloroethene, vinyl chloride     

Electrogenic cation transport across leech caecal epithelium

 Electrogenic cation transport across the caecal epithelium of the leech Hirudo medicinalis was investigated using modified Ussing chambers. Transepithelial resistance (R _T ) and potential difference (V _T ) were 61.0±3.5 Ω ⋅ cm² and −1.1±0.2 mV (n=149), respectively, indicating that leech caecal epithelium is a “leaky” epithelium. Under control conditions short circuit current (I _SC ) and transepithelial Na⁺ transport rate (I _Na ) averaged at 22.1±1.5 μA ⋅ cm^-2 and 49.7±2.6 μA ⋅ cm^-2, respectively. Mucosal application of amiloride (100 μmol ⋅ l^-1) or benzamil (50 μmol ⋅ l^-1) influenced neither I _SC nor I _Na . The transport system in the apical membrane showed no pronounced cation selectivity and a linear dependence on mucosal Na⁺ concentration. Removal of mucosal Ca²⁺ increased I _SC by about 50% due to an increase of transepithelial Na⁺ transport. Trivalent cations (La³⁺ and Tb³⁺, 1 mmol ⋅ l^-1 both) added to the mucosal Ringer solution reduced I _Na by more than 40%. Serosal ouabain (1 mmol ⋅ l^-1) almost halved I _SC and I _Na while 0.1% (=5.4 mmol ⋅ l^-1) DNP decreased I _Na to 11.8±5.1% of initial values. Serosal addition of cAMP increased both I _SC and I _Na whereas the neurotransmitters FMRFamide, acetylcholine, GABA, L-dopa, serotonin and dopamine failed to show any effects; octopamine, glycine and L-glutamate reduced I _Na markedly. On the basis of these results we conclude that in leech caecal epithelium apical uptake of monovalent cations is mediated by non-selective cation conductances which are sensitive to extracellular Ca²⁺ but insensitive to amiloride. Basolaterally Na⁺ is extruded via ouabain-sensitive and -insensitive ATPases. cAMP activates Na⁺ transport across leech caecal epithelium, although the physiological stimulus for cAMP-production remains unknown. Accepted: 20 May 1996     

Biomass, production and heterotrophic activity of bacterioplankton in the Great Astrolabe Reef lagoon (Fiji)

 Biomass, production and heterotrophic activity of bacterioplankton were determined for two weeks in the Great Astrolabe Reef lagoon, Fiji. Bacterial and Bacterial activities were distributed homogeneously throughout the water column (20 to 40 m deep) and varied little from site to site inside the lagoon. Bacterioplankton biomass and production also varied little over a diel period with coefficients of variation of 9 and 22%, respectively. On average, over the whole study, bacterial abundance was 0.77×10⁹ cells l^-1 and bacterial production averaged 0.36 μg-at. C l^-1 d^-1. Bacterial abundance and production were greater in the lagoon than in oceanic waters. Attachment to particles seems to provide an advantage for bacterioplankton growth because specific growth rates for attached bacterioplankton were, on average, significantly greater than that of the free community. Growth efficiency, determined by correlating the net increase of bacterial biomass and the net decrease of dissolved organic carbon (DOC) in dilution cultures, was very low (average 6.6%). Using carbon growth efficiency and bacterial production rates, heterotrophic activity was estimated to average 5.4 μg-at. C l^-1 d^-1. The turn-over rate of DOC (average 114 μg-at. C l^-1) due to bacterial consumption was estimated to be 0.048 d^-1 during the period of study. Accepted: 25 July 1998     

Tetrachloroethene metabolism of Dehalospirillum multivorans

Dehalospirillum multivorans is a strictly anaerobic bacterium that is able to dechlorinate tetrachloroethene (perchloroethylene; PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) as part of its energy metabolism. The present communication describes some features of the dechlorination reaction in growing cultures, cell suspensions, and cell extracts of D. multivorans. Cell suspensions catalyzed the reductive dechlorination of PCE with pyruvate as electron donor at specific rates of up to 150 nmol (chloride released) min^-1 (mg cell protein)^-1 (300 M PCE initially, pH 7.5, 25°C). The rate of dechlorination depended on the PCE concentration; concentrations higher than 300 M inhibited dehalogenation. The temperature optimum was between 25 and 30°C; the pH optimum at about 7.5. Dehalogenation was sensitive to potential alternative electron acceptors such as fumarate or sulfur; nitrate or sulfate had no significant effect on PCE reduction. Propyl iodide (50 M) almost completely inhibited the dehalogenation of PCE in cell suspensions. Cell extracts mediated the dehalogenation of PCE and of TCE with reduced methyl viologen as the electron donor at specific rates of up to 0.5 mol (chloride released) min^-1 (mg protein).^-1 An abiotic reductive dehalogenation could be excluded since cell extracts heated for 10 min at 95°C were inactive. The PCE dehalogenase was recovered in the soluble cell fraction after ultracentrifugation. The enzyme was not inactivated by oxygen.Abbreviations PCE Perchloroethylene or tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon - MV Methyl viologen     

Nitrogen and energy requirements of the short-tailed fruit bat (Carollia perspicillata): fruit bats are not nitrogen constrained

 Nitrogen (N) and energy (E) requirements were measured in adult Carollia perspicillata which were fed on four experimental diets. Bats ate 1.3–1.8 times their body mass ⋅ day^-1 and ingested 1339.5–1941.4 kJ ⋅ kg^-0.75 ⋅ day^-1. Despite a rapid transit time, dry matter digestibility and metabolizable E coefficient were high (83.3% and 82.4%, respectively), but true N digestibility was low (67.0%). Mass change was not correlated with E intake, indicating that bats adjusted their metabolic rate to maintain constant mass. Bats were able to maintain constant mass with digestible E intake as low as 1168.7 kJ ⋅ kg^-0.75 ⋅ day^-1 or 58.6 kJ ⋅ . Metabolic fecal N and endogenous urinary N losses were 0.87 mg N ⋅ g^-1 dry matter intake and 172.5 mg N ⋅ kg^-0.75 ⋅ day^-1, respectively, and bats required 442 mg N ⋅ kg^-0.75 ⋅ day^-1 (total nitrogen) or 292.8 mg N ⋅ kg^-0.75 ⋅ day^-1 (truly digestible nitrogen) for N balance. Based on E and N requirements and digestibilities, it was calculated that non-reproductive fruit bats were able to meet their N requirements without resorting to folivory and without over-ingesting energy. It was demonstrated that low metabolic fecal requirements allowed bats to survive on low-N diets. Accepted: 23 June 1996     

Denitrifying and methanogenic bacteria in the biofilm of a fixed-film reactor operated with methanol/nitrate demonstrated by immunofluorescence and microscopy

 A denitrifying bacterial biofilm population established on a polypropylene substratum of a fixed-film reactor was characterized by microscopy, scanning electron microscopy and immunofluorescence after 120 days of operation. The reactor, operated at pH 7.0, 22°C, and −180 mV with synthetic wastewater containing methanol/nitrate, achieved a denitrification rate of 0.24 mol NO^- ₃ l^-1 day^-1 with a removal efficiency for nitrate of 95%–99% at an organic loading rate of 0.325 mol methanol l^-1 day^-1. The gas produced contained 2%–3% (v/v) methane and 3%–4% (v/v) carbon dioxide in addition to nitrogen. The biofilm contained mainly cells of Methanobrevibacter arboriphilus antigenically related to strain DC, short, flagellated, gram-negatively staining rods of Pseudomonas sp. antigenically related to Pseudomonas stutzeri strain AN11, non-identified pink-pigmented rods and small lemon-shaped cells with mono- and bipolar appendages resembling prosthecate Hyphomicrobium sp. The biofilm analysis provided evidence for a syntrophy between the denitrifying, methylotrophic, bacterial consortium and hydrogenotrophic methanogens, which were identified by antigenic fingerprinting with 17 antibody probes. Received: 11 July 1994/Received revision: 23 September 1994/Accepted: 28 September 1994     

Octanoate increases cytosolic Ca2+ concentration and membrane conductance in ovine pancreatic acinar cells

 In order to investigate the cellular mechanisms involved in amylase release in response to stimulation with short-chain fatty acids, changes in intracellular calcium concentration ([Ca²⁺]_i), membrane current and amylase release were measured in pancreatic acinar cells of sheep. Both octanoate and acetylcholine raised [Ca²⁺]_i in acinar cells in a concentration-dependent manner. The rise in [Ca²⁺]_i in response to the stimulation with octanoate (10 mmol ⋅ l^-1) was reduced in a medium without CaCl₂, but was markedly enhanced by reintroduction of CaCl₂ into the medium up to 2.56 mmol ⋅ l^-1. Perfusion of the cells with a medium containing octanoate (5 mmol ⋅ l^-1) or acetylcholine (0.5 μmol ⋅ l^-1) immediately raised inward current across the cell membrane at a holding-membrane potential of −30 mV. The inward current became greater as the holding potential became more negative. The equilibrium potential was 1.8 mV and 3.9 mV for octanoate and acetylcholine, respectively, being consistent with that for Cl^-. Although intracellular application of octanoate through a patch-clamp pipette also raised inward current after several minutes in some cells (4 out of 12), this possibility was significantly smaller than that for extracellular application. In other cells, even though the intracellular application of octanoate did not cause an increase in current, it always caused responses immediately after introduction of the fatty acid into the medium. Stimulation with fatty acid as well as acetylcholine raised amylase release in a concentration-dependent manner in cells dispersed from tissue segments with crude collagenase and trypsin inhibitor. Without trypsin inhibitor, crude collagenase significantly and selectively reduced the octanoate (10 mmol ⋅ l^-1)-induced amylase release. Dispersion with crude collagenase and trypsin significantly reduced both responses induced by octanoate and acetylcholine (5.5 μmol ⋅ l^-1). We conclude that fatty acids and acetylcholine increase [Ca²⁺]_i, which consequently evokes a rise in transmembrane ion (Cl^-) conductance and amylase release, and that trypsin-sensitive protein(s) in the cell membrane are involved in secretory processes activated by stimulation with fatty acids in ovine pancreatic acinar cells. Accepted: 14 May 1996     

Sulfated extracellular polysaccharide production by the halophilic cyanobacterium Aphanocapsa halophytia immobilized on light-diffusing optical fibers

 The cyanobacterium, Aphanocapsa halo-phytia MN-11, was immobilized in calcium alginate gel and coated on light-diffusing optical fibers (LDOF) for sulfated extracellular polysaccharide production. Results indicated that sulfated extracellular polysaccharide production depends on the number of immobilized cells and the light intensity. In addition, the production rate reached 116.0 mg (mg dry cells)^-1 day^-1 when the cells that were immobilized on LDOF were incubated under a light intensity of 1380 cd sr m^-2 at a cell concentration of 1.0×10⁸ cells/cm³ gel. Cells immobilized on LDOF produced about ten times more sulfated extracellular polysaccharide than those immobilized in calcium alginate beads only (11.7 mg(mg dry cells)^-1day^-1). Received: 31 March 1995/Revised last revision 12 June 1995/Accepted 26 July 1995     

Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater

Denitrification of a high-strength synthetic wastewater (150 g NO^- ₃ l^-1) was carried out using a wine distillery effluent as an example of an industrial carbon source (22.7 g chemical oxygen demand l^-1). Two configurations were tested: one consisted of an acidogenesis reactor followed by a denitrifying reactor and the other was a single reactor directly fed with the raw effluents. In both cases, denitrification was achieved at a nitrate load of 9.54 g NO^- ₃ l^-1 day^-1 (2.19 g N as NO^- ₃ l^-1 day^-1) with good specific reduction rates: 32.6 mg and 35.2 mg N as NO _x  g volatile suspended solids h^-1, calculated on a single day, for the two-step and the one-step process respectively. Dissimilatory nitrate reduction to ammonium did not occur, even in the one-step process. Received: 26 October 1995/Received revision: 15 February 1996/Accepted: 20 February 1996