Ethoxyresorufin O-deethylase (EROD) activity in the liver of dab (Limanda limanda L.) and flounder (Platichthys flesus L.) from the German Bight. EROD expression and tissue contamination

Ethoxyresorufin O-deethylase (EROD) activity was measured in the liver of dab (Limanda limanda) and flounder (Platichthys flesus) from the German Bight (southern North Sea) and compared with muscle and liver polychlorinated biphenyl (PCB) concentrations in an attempt to relate EROD activity to PCB body burden. In none of the different datasets (species-, tissue- or matrix-dependent) was a significant (P<0.05) correlation between PCB tissue contamination and EROD activity found. Yet EROD activity was significantly correlated with polycyclic aromatic hydrocarbons (PAH) levels (phenanthrene, fluoranthene, pyrene) in muscle tissue, indicating a possible dependence of EROD expression on other ubiquitous organic contaminants, thus making it a suitable biomarker for general pollution. Received: 14 April 1999 / Received in revised form: 10 July 1999 / Accepted: 15 July 1999     

The use of fish metabolic, pathological and parasitological indices in pollution monitoring

  An integrated biological effect monitoring concept has been tested in flounder (Platichthys flesus L.) from four locations with different anthropogenic impact in the German Bight. During 3 years of sampling, biomarkers at all levels of biological organisation from the molecular to the ecosystem level were applied and tested on 742 individual fish of body lengths between 18 and 25 cm. At the ecosystem level, the fish were taken as a habitat for the parasite assemblage. The hypothesis was that changes in the environment might lead to changes in the species diversity of parasites and in the infection intensity of single species, as well as between heteroxenic and monoxenic parasite species (H/M ratio). At the molecular level, activity of the CYP1A-dependent monooxygenase ethoxyresorufin O-deethylase (EROD) was used as a biomarker of exposure. At the subcellular level, the integrity of lysosomal membranes in hepatocytes was taken as an indicator of non-specific acute and chronic toxic effects. Both biomarkers are recommended by the ICES Advisory Committee on the Marine Environment for the application in biological effects monitoring programmes. In addition, neutral lipid content in the liver was used as a marker for pathologically induced fat accumulation. In the same individual fish, a new method for the measurement of macrophage aggregate activity in the liver was tested for its application and reliability in reflecting immunosuppression. Tests were accompanied by chemical analysis of standard organochlorine and heavy metal residues in flounder tissue. A total of 33 parasite species were found. As an indicator species, the mean abundance of Trichodina sp. reflected best the pollution gradient observed with highest infection intensity at the most polluted location. Species diversity was significantly higher in fish caught near the reference site and significantly lower in fish from the polluted Elbe estuary. The use of the heteroxenous/monoxenous species ratio as a marker was not useful at the locations investigated because of the dominance of heteroxenous species at all habitats. Since EROD activity and macrophage aggregate activity were dependent on sex and maturity of female flounder, only male fish were taken into consideration for the integrated evaluation of data. All biochemical and histochemical tests were able to reflect accurately the site-specific differences, as well as an observed pollution event at the end of 1995 as determined by chemical analyses. The correlation analysis revealed a connection not only between the single parasitological and biochemical parameters but also within these groups. The non-specific immune response and Trichodina infection intensity were correlated with all other parameters, leading to the assumption that these may serve as links between the lowest and the highest levels of biological organisation. The simultaneous use of metabolic and parasitological results facilitated the interpretation of the observed variations of the data and the distinction between natural variation and pollution-mediated effects. Received: 1 March 1999 / Received in revised form: 25 May 1999 / Accepted: 7 June 1999     

Multivariate statistical approach to the temporal and spatial patterns of selected bioindicators observed in the North Sea during the years 1995–1997

A comprehensive database, containing biological and chemical information, collected in the framework of the bilateral interdisciplinary MARS project (”biological indicators of natural and man-made changes in marine and coastal waters”) during the years 1995–1997 in the coastal environment of the North Sea, was subjected to a multivariate statistical evaluation. The MARS project was designated to combine a variety of approaches and to develop a set of methods for the employment of biological indicators in pollution monitoring and environmental quality assessment. In total, nine ship cruises to four coastal sampling sites were conducted; 765 fish and 384 mussel samples were analysed for biological and chemical parameters. Additional information on the chemical background at the sampling sites was derived from sediment samples, collected at each of the four sampling sites. Based on the available chemical data in sediments and black mussel (Mytilus edulis) a pollution gradient between the selected sites, was established. The chemical body burden of flounder (Platichthys flesus) from these sites, though, did not reflect this gradient equally clear. In contrast, the biological information derived from measurements in fish samples displayed significant a regional as well as a temporal pattern. A multivariate bioindicator data matrix was evaluated employing a factor analysis model to identify relations between selected biological indicators, and to improve the understanding of a regional and temporal component in the parameter response. In a second approach, applying the k-means algorithm on the data matrix, two significantly different clusters of samples, characterised by the current health status of the fish, were extracted. Using this classification a temporal, and in the second order, a less pronounced spatial effect was evident. In particular, during July 1996, a clear sign of deteriorating environmental conditions was extracted from the biological data matrix. Received: 20 June 1999 / Received in revised form: 8 November 1999 / Accepted: 8 November 1999     

A gene-based RFLP map of petunia

Due in large part to the data accumulated from years of classic genetic analysis, petunia (Petunia hybrida Vilm) has remained a useful model system, particularly for studies of gene regulation and genome structure. We have used three segregating populations of petunia, including those serving as the source of an earlier actin gene RFLP map, for RFLP mapping of several additional genes. Twenty-seven loci have been merged with 11 previously mapped morphological and biochemical markers. Our results contribute additional evidence to reports of a high degree of genome plasticity and segregation distortion in this species and suggest that petunia may be a useful plant system for detailed analysis of plant genome organization, activity and evolution. Received 14 July 1999 / Accepted: 30 July 1999     

Isolation and characterisation of a ropy Lactobacillus strain producing the exopolysaccharide kefiran

A capsular-polysaccharide-producing strain, LM-17, was isolated from kefir grains and was identified as a slime-forming, rod-shaped Lactobacillus. According to ¹H- and ¹³C-NMR spectral data, the exopolysaccharide produced by the isolated bacterial strain is identical to the glucogalactan extracted from kefir grains and therefore known as kefiran. The kefiran produced was characterised by means of viscosity, optical rotatory power, circular dichroism and IR spectral measurements. A batch procedure was set up for the culture and extraction of the exopolysaccharide in laboratory conditions, resulting in a yield of 2 g/l purified kefiran from the culture supernatant of the LM-17 strain. Received: 6 April 1999 / Received revision: 30 July 1999 / Accepted: 13 August 1999     

News & Notes: Detection of Microorganisms with Overall Cellulolytic Activity

A modification is described of the plate method for the detection of microorganisms with overall cellulolytic activity, including those like Cytophaga, in which the activity is cell bound. Within a few days of incubation colonies of cellulose-degrading bacteria formed holes in discs of lens paper placed on freshly inoculated agar plates. Received: 14 June 1999 / Accepted: 20 July 1999     

Biosynthesis of citric acid by Yarrowia lipolytica repeat-batch culture on ethanol

After analysis of batch culture and identification of the ways for prolongation of citric acid active synthesis by yeast, repeat-batch (RB) cultivation was suggested. Yarrowia lipolytica strain RB cultivation was studied and optimal conditions for cultivation selected. It was shown that when applying RB cultivation, better results were obtained than for batch cultivation. The activity of the culture remained stable after cultivation for more than 700 h. Comparative analysis of enzyme activities confirmed the regularity of the effect described, as the activity of practically of all the enzymes participating in ethanol oxidation and citric acid biosynthesis remained stable over time during RB cultivation. Advantages of RB cultivation for the production of citric acid by yeast are discussed. Received: 1 March 1999 / Received revision: 28 June 1999 / Accepted: 5 July 1999     

Influence of a synbiotic mixture consisting of Lactobacillus acidophilus 74-2 and a fructooligosaccharide preparation on the microbial ecology sustained in a simulation of the human intestinal microbial ecosystem (SHIME reactor)

 Lactobacillus acidophilus 74-2, which is used in probiotic products, was administered, with fructooligosaccharide in a milk-based product, to the second vessel (duodenum/jejunum) of the SHIME reactor, an in vitro simulation of the human intestinal microbial ecology. The main focus of this study was to monitor the changes of the population density of selected bacterial species in the intestine and the changes of metabolic activities during the supplementation of L. acidophilus and fructooligosaccharide in the SHIME reactor. Interestingly, the addition of L. acidophilus 74-2 with fructooligosaccharide gave rise to an increase of bifidobacteria. Moreover, major positive changes occurred in the production of volatile fatty acids: a strong upward trend was observed especially in the case of butyric acid and propionic acid. Furthermore a noticeable increase of β-galactosidase activity was monitored, while the activity of β-glucuronidase, generally considered undesirable, declined. Received: 22 January 1999 / Received revision: 13 July 1999 / Accepted: 13 August 1999     

Honesty of agonistic signalling and effects of size and motivation asymmetry in contests

Game theoretical models predict that the main function of fighting behaviour is to assess the relative fighting ability of opponents. The sequential assessment game has often been used to investigate contests, while honest signalling theory has received much less attention. With the wolf spider Hygrolycosa rubrofasciata we investigated whether male agonistic signalling can reveal honest information about fighting ability, and how size and motivation asymmetries affect male fighting behaviour. We also determined whether male–male competition affects the courtship behaviour of the males. We found that agonistic drumming activity is an honest indicator of male fighting ability, and that relative size asymmetry and motivation to fight both contribute to the fighting ability. We also found that male–male competition decreases the courtship drumming rate of subdominant males, suggesting that male–male competition limits the opportunities for female choice. We conclude that sequential assessment and honest signalling may both be used in settling contests, and that they may be used simultaneously. Received: 10 December 1998 / Received in revised form: 23 June 1999 / Accepted: 5 July 1999     

Oxygen-dependent xylitol metabolism in Pichia stipitis

Pichia stipitis CBS 6054 was cultivated in chemostat cultures under aerobic and oxygen-limited conditions with xylitol alone, a mixture of xylitol and glucose and a mixture of xylitol and xylose. Xylitol metabolism was strictly respiratory and no ethanol was formed. Simultaneous feeding of xylitol and glucose and xylitol and xylose to oxygen-limited xylitol-pregrown cells resulted in ethanol formation. In vitro both pyruvate decarboxylase activity and alcohol dehydrogenase activity were present in cells metabolising xylitol under oxygen-limited conditions; however, this did not result in ethanol formation. Glucose, xylose and xylitol utilisation, respectively, were compared under anaerobic conditions with regard to growth rate, carbon source and oxygenation level during pre-cultivation. Irrespective of pre-growth conditions, xylitol was not metabolised under anaerobic conditions, whereas ethanol was formed from both xylose and glucose. Anaerobic xylose utilisation required induction of a xylose-utilising metabolic pathway during pre-cultivation. Received: 23 February 1999 / Received last revision: 20 July 1999 / Accepted: 1 August 1999     

The effects of the polychaete Nereis diversicolor on the distribution and transplanting success of Zostera noltii

Loss of saltmarsh vegetation in south-east England is a significant problem for conservation and coastal defence. The losses of vegetation began in the 1930s with the loss of intertidal Zostera marina and have continued more recently. Some preliminary trials at re-establishing Zostera in some estuaries of Essex have not been successful. This paper addresses the hypothesis that the infauna, particularly the polychaete Nereis diversicolor, may restrict natural colonisation by Zostera and reduce the success of transplanting trials. In field experiments, Z. noltii were transplanted into areas where Nereis were common, close to an established seagrass bed and into two other estuaries. The transplants protected from the effects of the polychaetes by netting had a higher survival rate, lower index of root damage and greater biomass at the end of the experiments than those that were unprotected. In laboratory experiments, Nereis reduced the survival of Z. noltii. They were observed grasping the leaves and pulling them into their burrows. These results indicate that herbivory and disturbance by N. diversicolor is responsible, at least in part, for the restriction of the distribution of Z. noltii and may have been important in limiting the success of previous transplanting experiments. A hypothesis is proposed which states that there are two stable states on the upper mud-flats. One state is dominated by plants, including Zostera spp., which prevent colonisation by burrowing infauna, and the other is dominated by infauna which prevent colonisation by plants. Managing these two states could be the key to re-establishing the early successional stages of saltmarsh development. Received: 15 June 1999 / Received in revised form: 6 July 1999 / Accepted: 8 July 1999     

Simulation of photosynthetic plasticity in response to highly fluctuating light: an empirical model integrating dynamic photosynthetic induction and capacity

An empirical model was developed to simulate photosynthetic responses of leaves to highly fluctuating light, with a special focus on the functional role of photosynthetic induction and capacity. Based on diurnal courses of light as input data, which were recorded at natural plant sites, we applied this model to simulate the corresponding course of net photosynthesis (output data) for leaves of two neotropical tree species. All six model input parameters (leaf-specific) were obtained via measurements of leaf gas exchange. The model was tested for leaves in their natural environments, characterized by frequent light-flecks. We compared measured carbon gains with computed ones, using a standard steady-state and our induction model. Simulation runs with the steady-state model can result in an immense overestimation of the true situation, by 13.4% at open sites [pioneer species Heliocarpus appendiculatus (Turczaninow)] and by 86.5% at low light environments of the understorey [mid to late successional species Billia colombiana (Planchon and Lindley)]. These significant overestimations, particularly in the understorey, are mainly the consequence of neglecting a dynamic photosynthetic induction under fluctuating light conditions. The model presented here resulted in clearly improved predictions; in open and understorey sites the true carbon gain of leaves was computed with a mean error of less than 7%. As most leaves at natural plant sites are exposed to light environments allowing for dynamic rather than steady-state CO₂ assimilation, the significance of such induction models is evident and is discussed in relation to scaling-up from leaf to canopy and to the whole plant indicating a large potential for errors. Received: 3 May 1999 / Accepted: 9 July 1999     

Keratinase of Doratomyces microsporus

 The fungus Doratomyces microsporus produced an extracellular keratinase during submerged aerobic cultivation in a medium containing a protein inducer for enzyme synthesis. The keratinase was purified to homogeneity using hydrophobic interaction chromatography followed by gel chromatography. The molecular weight was estimated to be 33 kDa (from SDS-PAGE analysis) or 30 kDa (by gel chromatography), suggesting a monomeric structure. The isoelectric point of the enzyme was determined to be around 9. The optimal pH and temperature for keratinolytic activity were pH 8–9 and 50 °C, respectively. The serine protease inhibitor PMSF totally inhibited the keratinase. The enzyme was not glycosylated. It was capable of hydrolysing different keratinous materials as well as some non-keratinous proteins. Hydrolysis of some synthetic substrates, specific for known proteinases, suggested that the keratinase of D. microsporus is close to proteinase K. Received: 9 July 1999 / Received revision: 13 September 1999 / Accepted: 17 September 1999     

Identification of interacting mixed cultures of lactic acid bacteria by their exclusion from a model predicting the acidifying activity of non-interacting mixed cultures

A model predicting the acidifying activity of mixed cultures of lactic acid bacteria and based on the lack of interaction between the strains has been investigated to identify interacting cultures. Three mixed cultures with Streptococcus thermophilus TH3 and ST7 and Lactobacillus delbrueckii ssp. bulgaricus LB10 were grown on milk. The acidifying activities of the two mixed cultures TH3/LB10 and TH3/ST7 were predicted accurately by the model, with mean prediction errors of 7.7% and 14.1%, respectively. However, the model underestimated the acidifying activity of the mixed culture ST7/LB10, with a mean prediction error of 43.5%, which provides evidence of positive interaction between the strains ST7 and LB10 during acidification. Received: 26 April 1999 / Received revision: 16 June 2000 / Accepted: 18 June 2000     

Reduction of aryl acids by white-rot fungi for the biocatalytic production of aryl aldehydes and alcohols

Ligninolytic basidiomycetes were screened for their ability to reduce aryl acids to the corresponding aldehydes and alcohols. Seven fungal strains converted p-anisic acid in high molar yields to the reduced products. The white-rot fungus Bjerkandera sp. strain BOS55 was one of the best reducing strains and was highly tolerant towards high concentrations of different aromatic acids. It was tested for the reduction of p-anisic, veratric, 3-chloro-4-methoxybenzoic, 3,5-dichloro-4-methoxybenzoic, 3,4-dichlorobenzoic, 4-fluorobenzoic, and 3-nitrobenzoic acids. All of these compounds were reduced to their corresponding aldehydes and alcohols. Received: 22 March 1999 / Received revision: 12 July 1999 / Accepted: 1 August 1999     

Study on the production of 6-pentyl-α-pyrone using two methods of fermentation

 The lactone 6-pentyl-α-pyrone has a characteristic coconut aroma and is produced by Trichoderma species. A study on the fermentative production of 6-pentyl-α-pyrone in both surface and submerged conditions by Trichoderma harzianum was carried out. Maximum concentrations of 455 mg/l and 167 mg/l after 96 h and 48 h of fermentation in surface and submerged conditions, respectively, were obtained without using any additional recovery operations. The resultant yields are higher than those previously reported in the literature, which may be attributable to strain characteristics in combination with the choice of fermentation conditions employed in the present study. Enough scope exists for further improvement in the yields by optimizing the cultural and nutritional parameters. Received: 13 July 1999 / Received revision: 19 November 1999 / Accepted: 19 November 1999     

Inhibition of fungal growth in planta and in vitro by transgenic tobacco expressing a bacterial nonheme chloroperoxidase gene

 Transgenic tobacco plants producing chloroperoxidase (CPO-P), encoded by a novel gene from Pseudomonas pyrrocinia, were obtained by Agrobacterium-mediated transformation. Successful transformation was shown by PCR, Southern, northern and western blot analyses, and assays of CPO-P enzyme activity. Extracts from plants transformed with the CPO-P gene significantly reduced Aspergillus flavus colonies by up to 100% compared with extracts from control plants transformed with pBI121. Compared with controls, the transformed plants showed increased disease resistance in planta against a fungal pathogen, Colletotrichum destructivum, the causal agent of tobacco anthracnose. Received: 10 March 1999 / Revision received: 22 June 1999 · Accepted: 5 July 1999     

Lessons learned from Sphingomonas species that degrade abietane triterpenoids

Abietane terpenoid-degrading organisms include Sphingomonas spp which inhabit natural environments and biological treatment systems. An isolate from the high Arctic indicates that these organisms occur far from trees which synthesize abietanes and suggests that some of these organisms can occupy a niche in hydrocarbon-degrading soil communities. Abietane-degrading Sphingomonas spp provide additional evidence that the phylogeny of this genus is independent of the catabolic capabilities of its members. Studies of Sphingomonas sp DhA-33 demonstrate that biological treatment systems for pulp mill effluents have the potential to mineralize abietane resin acids. On the other hand, these studies indicate that some chlorinated dehydroabietic acids are quite recalcitrant. Strain DhA-33 grows relatively well on some chlorinated dehydroabietic acids but transforms others to stable metabolites. Using strain DhA-33, a novel method was developed to measure the metabolic activity of an individual population within a complex microbial community. Oligonucleotide hybridization probes were used to assay the 16S rRNA:rDNA ratio of DhA-33 as it grew in an activated sludge community. However, this method proved not to be sufficiently sensitive to measure naturally occurring resin acid-degrading populations. We propose that the same approach can be modified to use more sensitive assays. Received 01 May 1999/ Accepted in revised form 19 July 1999     

An extractive membrane biofilm reactor for degradation of 1,3-dichloropropene in industrial waste water

A bacterial biofilm, capable of mineralising a technical mixture of cis- and trans-1,3-dichloropropene (DCPE), was enriched on the biomedium side of an extractive membrane biofilm reactor (EMBR). The membrane separates the biomedium from the industrial waste water, in terms of pH, ionic strength and the concentration of toxic chemicals. The biofilm, attached to a silicone membrane, is able to mineralise DCPE after its diffusion through the membrane. Five bacterial strains with degradation capabilities were isolated from the metabolically active biofilm and further investigated in batch experiments. Two of them, Rhodococcus erythropolis strains EK2 and EK5, can grow with DCPE as the sole carbon source. Pseudomonas sp. EK1 utilises cis-3-chloroallylalcohol and cis-3-chloroacrylic acid, whereas the metabolite trans-3-chloroacrylic acid represents a dead-end product of the pathway of this strain. The other two strains, Delftia sp. EK3 and EK4, although unable to grow with DCPE as the carbon source, can transform DCPE and its upper-pathway intermediates at reasonable conversion rates. They may represent helper functions of the biofilm consortium, which mineralised up to 12.5 mmol DCPE per hour per gram of biomass protein. Higher feed rates in the EMBR (up to 15 mmol per hour per 100-l bioreactor volume) and shock loads corresponding to concentrations up to 1.8 mmol l⁻¹ led to a significant increase in the freely floating bacterial biomass in the reactor medium (OD₅₄₆= 0.2). At the standard operating feed rate of 1.8 mmol h⁻¹, the free biomass concentration was very low (OD₅₄₆= 0.04). Received: 23 April 1999 / Received revision: 1 July 1999 / Accepted: 5 July 1999     

Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy)

Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.