Porous biocarrier-enhanced biodegradation of crude oil contaminated soil

Soil contamination with crude oil from petrochemicals and oil exploitation is an important worldwide issue. Comparing available remediation techniques, bioremediation is widely considered to be a cost-effective choice; however, slow degradation of crude oil is a common problem due to the low numbers of bacteria capable of degrading petroleum hydrocarbons and the low bioavailability of contaminants in soil. To promote crude oil removal, biocarrier for immobilization of indigenous hydrocarbon-degrading bacteria was developed using porous materials such as activated carbon and zeolite. Microbial biomass reached 10¹⁰ cells g^?1 on activated carbon and 10⁶ cells g^?1 on zeolite. Total microbial and dehydrogenase activities were approximately 12 times and 3 times higher, respectively, in activated carbon than in zeolite. High microbial colonization by spherical and rod shapes were observed for the 5–20 μm thick biofilm on the outer surface of both biocarriers using electronic microscopy. Based on batch-scale experiments containing free-living bacterial cultures and activated carbon biocarrier into crude oil contaminated soil, biocarrier enhanced the biodegradation of crude oil, with 48.89% removal, compared to natural attenuation with 13.0% removal, biostimulation (nutrient supplement only) with 26.3% removal, and bioaugmentation (free-living bacteria) with 37.4% removal. In addition, the biocarrier increased the bacterial population to 10⁸ cells g^?1 dry soil and total microbial activity to 3.5 A₄₉₀. A hypothesis model was proposed to explain the mechanism: the biocarrier improved the oxygen, nutrient mass transfer and water holding capacity of the soil, which were the limiting factors for biodegradation of non-aqueous phase liquid (NAPL) contaminants such as crude oil in soil.Scientific relevanceThis study explored the role of biocarrier in enhancing biodegradation of hydrophobic contaminants such as crude oil, and discussed the function of biocarrier in improving oxygen mass transfer and soil water holding capacity, etc.     

Bioaugmentation of carbofuran by Burkholderia cepacia PCL3 in a bioslurry phase sequencing batch reactor

The effectiveness of bioremediation technology in the removal of carbofuran from contaminated soil using a bioslurry phase sequencing batch reactor (SBR) was investigated. A 2-L laboratory glass bottle was used as a bioreactor with a working volume of 1.5 L at room temperature (27 ± 2 °C). One total cycle period of the SBR was comprised of 1 h of fill phase, 82 h of react phase, and 1 h of decant phase. The carbofuran concentration in the soil was 20 mg/kg soil. A carbofuran degrader isolated from carbofuran phytoremediated soil, namely Burkholderia cepacia PCL3 (PCL3) immobilized on corncob, was used as the inoculum. The results revealed that bioaugmentation treatment (addition of PCL3) gave the highest percentage of carbofuran removal (96.97%), followed by bioaugmentation together with biostimulation (addition of molasses) treatment (88.23%), suggesting that bioremediation was an effective technology for removing carbofuran in contaminated soil. Abiotic experiments, i.e. autoclaved soil slurry with corncob and no PCL3 treatment and autoclaved soil slurry with no PCL3 treatment, could adsorb 31.86% and 7.70% of carbofuran, respectively, which implied that soil and corncob could act as sorbents for the removal of carbofuran.     

Biological indices of toxicity in tropical legumes grown in oil-contaminated soil

This study evaluated the toxic effects of total petroleum hydrocarbons (TPH) on growth of the legumes Crotalaria incana L. and Leucaena leucocephala Lam., and on the development of nitrogen-fixing soil microorganisms, using biological toxicity indices and the toxicity potential index (TPI_c), which enable comparison of effects of exposure time and concentration. Growth and biomass accumulation in both plant species decreased with high pollutant concentrations. The EC₅₀ and the NOEC were not identified for either species. The Phytotoxicity Relative Index showed that root length was most strongly affected by the oil, and the Impact Index on Nitrogen Fixer Microorganisms indicated that, despite damage to the root system, L. leucocephala rhizosphere bacteria doubled at 10,000 mg kg⁻¹ TPH after of 240 days of exposure. Finally, the TPI_c revealed that C. incana was more sensitive than L. leucocephala to chronic TPH toxicity and might strongly depend on beneficial soil bacteria.     

Bioremediation of phenol-contaminated water and soil using magnetic polymer beads

In order to easily separate pollutant-absorbing polymer beads from contaminated soil or water, novel polymer beads containing magnetic particles were developed. The polymer beads containing 4.67% (w/w) magnetic particles exhibited an almost identical partitioning coefficient for phenol compared to that of the pure polymer. A 1.5 L phenol solution of 2000 mg/L added to a bioreactor was reduced to 481 mg/L phenol within 3 h by adding 100 g of these magnetic beads, and the phenol was completely degraded by microorganisms in 16 h. The magnetized beads were then readily removed from the bioreactor by a magnet with 10,000 G, and subsequently detached for re-use. 500 g of soil contaminated with 4 mg-phenol/g-soil was also contacted with 100 g beads, and greater than 97% removal of phenol from the soil was achieved within 1 day. The phenol-absorbing beads were easily separated from the soil by the magnet and transferred into a fermentor. The phenol was released from the beads and was degraded by the microorganism in 10 h. Modifying polymers to possess magnetic properties has greatly improved the ease of handling of these sequestering materials when decontaminating soil and water sources, in conjunction with contaminant release in partitioning bioreactors.     

Chemical and genetic diversity of two Mediterranean subspecies of Teucrium polium L.

Chemical and genetic diversity of Teucrium polium L. subsp. polium from western Algeria and T. polium L. subsp. capitatum from Corsica were investigated. Diversity within and among the two populations of subspecies was assessed according to the chemical composition of their essential oils and the genetic diversity. Chemical analysis was performed using a combination of capillary GC-RI and GC/MS after fractionation using column chromatography. Genetic structures were mapped using three polymorphic genetic markers: two chloroplast markers (RPL32-TRNL and TRNL-F) and ribosomal nuclear markers (ITS region). The statistical analysis showed that both subspecies were clearly distinguished by these chemical and genetic markers. The oil chemical compositions differed qualitatively and quantitatively between the subspecies. Both collective oils were dominated by hydrocarbon compounds however the Algerian sample oils exhibited higher amounts of hydrocarbon sesquiterpenes than those of Corsica (31.2 g/100 g vs. 4.4 g/100 g) while the latter displayed higher amounts of hydrocarbon monoterpenes than the first (59.3 g/100 g vs. 34.3 g/100 g). Neighbor-joining, Maximum likelihood and Bayesian trees constructed from chloroplast markers and nuclear ITS region sequences showed the existence of two groups associated with taxonomic and chemical characteristics. The study indicated that variation in the essential oil composition within subspecies depends on genetic background. The samples of subsp. capitatum from Corsica are a homogeneous group, in contrast to samples of subsp. polium from Algeria which were clustered in two groups. Chemical and genetic diversity of Algerian populations could be explained by geographical isolation of the populations. In addition, the morphological polymorphism observed throughout the colour of flowers could be explained by environmental parameters as well as the soil pH.     

Assessment of hydrocarbon biodegradability in clayed and weathered polluted soils

Hydrocarbon biodegradation in clayed and weathered polluted soils is a challenge; thus the aim of the present study was to determine the best experimental conditions that improve the hydrocarbon biodegradability in clayed and weathered polluted soils, modifying the nitrogen and phosphorous content considering the C:N:P ratio and the water content as a percentage of the water-holding capacity of the soil. Biodegradation tests were performed in microcosms containing 20 g of dry soil at 30 °C. A uniform-precision central composite design of second order with three levels was used to assess the effect of nutrient and water content adjustment on the hydrocarbon degradation rate, total carbon consumption, and microbial activity. The results showed that the water-holding capacity corresponding to 350% and a C:N:P ratio of 100:7.5:0.66 were the best experimental conditions for obtaining the highest hydrocarbon degradation rate (1145 mg TPH kg^?1 dry soil day^?1), whereas the hydrocarbon degradation rate in a non-stimulated control was only 129 mg TPH kg^?1 dry soil day^?1. Water content was the factor that showed the highest significant effect (p ≤ 0.05) on the hydrocarbon degradation rate. The results of the present study allowed the achievement of the best experimental conditions that enhance hydrocarbon biodegradability in clayed and weathered polluted soils. Also, these conditions are proposed for use as a biodegradability assay.     

Seasonal dynamics of soil fauna in the subalpine and alpine forests of west Sichuan at different altitudes

The climate (especially temperature) often plays an important role in the structure, function as well as composition of soil organisms in different latitudes and altitudes. As one of the essential components of soil ecosystem, soil faunal community not only lays their roles as soil engineer in material cycling and energy flow, but also acts as the sensitive bio-indicator to environmental change. However, little information has been available on the responses of soil faunal community to the changed environment at different altitudes and seasons. In order to understand the seasonal dynamics of soil faunal diversity under different forests with varying altitudes, three fir (Abies faxoniana) forests were selected covering a 600 m vertical transition zone. The primary fir forest at 3600 m (A1) of altitude, mixed fir and birch forest at 3300 m (A2) of altitude, and secondary fir forest at 3000 m (A3) of altitude are representative forests in the subalpine and alpine region of west Sichuan. A 2 years study was conducted in the three subalpine and alpine forests from May in 2009 until October in 2010. Soil samples were collected in both the soil organic layer and mineral soil layer. Soil macro-fauna were picked up by hand in the fields. Meso/micro-fauna and damp living fauna were separated and collected from the soil samples by Baermann and Tullgren methods in laboratory, respectively. A total of 74,827 individuals were collected in the 2 years, belonging to seven phyla, 16 classes, 31 orders and 125 families by preliminary identification. Similar dominant groups were detected in different forests at different altitudes, consisting of Spirostreptida, Formicidae, Staphylinidae, Hesperinidae, Onychiuridae, Isotomidae, Oribatuloidae, Alicoragiidae, Secernentea, and Adenophorea. In contrast, the ordinary species of macro-fauna and the ratios of Acarina to Collembolan were obviously different. For instance, the ordinary species were dominated by Cydmaenidae and Mycetophilidae at the A1, Scaphidiidae and Helicinidae at the A2, and Lumbricida and Agelenidae at the A3, respectively. Both the individual density and the number of soil faunal groups were significantly higher in soil organic layer than those in mineral soil layer. The density and group of macro-, meso- and micro-fauna in different forests showed the order as A2 > A1 > A3, but the density of damp living fauna showed the order as A1 > A2 > A3. The functional groups of macro-fauna were mainly dominated by saprozoic. The highest density and group of macro-fauna was observed in August, while the highest value of meso/micro-fauna was detected in October. In addition, the Jacard similarity indices showed that the composition and structure of soil fauna were similar in the different forests varied with altitudes, but the Shannon–Wiener indices were significantly different. The highest values of Shannon–Wiener indices were observed in October at both the A1 and A3, and in August at the A2. The results suggested that soil faunal community kept a high diversity in the subalpine and alpine forests of west Sichuan, and their structures were significantly affected by the variation of altitudes, which provided important scientific evidences for understanding the ecological processes in the subalpine and alpine coniferous forests.     

Role and characteristics of problematic biofilms within the removal and mobility of trace metals in a pilot-scale membrane bioreactor

The characteristics of problematic biofilms (i.e., fouling and clogging layers) were studied with regards to the removal and fate of trace metals (contents well under 100 μg/L) during the long-term operation of a pilot-scale membrane bioreactor for the treatment of real wastewaters from a large industrial area.Results showed that clogging layer was more effective than suspended activated sludge in the biosorption of As > Zn > Ni > Cd > Sb > Fe > Se due to the synergic effects of extracellular polymeric compounds and metal-resistant bacteria. In fact the selective microbial speciation of the phylum of Bacteroidetes, which is highly resistant to heavy metals, was observed in the clogging sludge in spite of the very low concentration of dissolved metals in the bioreactor.Compared to the suspended activated sludge, the clogging layer enhanced the biosorption of very toxic substances such as As, Cd and Ni. In fact, the metal contents were respectively: 7.9–7.4 vs. 690–840 μgAs/kgTS; 1.5–2.2 vs. 149–219 μgCd/kgTS; 58.8–71.7 vs. 227–298 μgNi/kgTS. Then, the potential desorption of metals during the membrane acid cleanings was estimated as relevant as 10–15% of the metals associated to the clogging sludge. The combined effects of pH and the selected microbial community, and the minor effect of the redox potential, let us conclude on the major importance of bio-sorption/desorption mechanisms with respect to bio-precipitation/dissolution.     

Degradation of 2,4-dichlorophenol by Bacillus sp. isolated from an aeration pond in the Baikalsk pulp and paper mill (Russia)

A pure culture of 2,4-dichlorophenol (2,4-DCP)-degrading bacteria was isolated from a natural enrichment that had been adapted to chlorophenols in the aeration pond of the Baikalsk pulp and paper mill (Russia). The bacteria were identified by 16S rDNA intergenic region analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (1545 bp) in the GenBank database revealed that these bacteria are related to Bacillus cereus GN1. Degradation of 2,4-DCP was studied using this culture in liquid medium under aerobic conditions, at initial concentrations of 20–560 μM 2,4-DCP. The 2,4-DCP degradation rates by B. cereus GN1 could be determined at concentrations up to 400 μM. However, higher concentrations of 2,4-DCP (560 μM) were inhibitory to cell growth.     

Biological treatment of oilfield-produced water: A field pilot study

A field test was conducted on a hydrolysis acidification/bio-contact oxidation system (HA/BCO) to treat oilfield-produced water with high salinity. By operating the biodegradation system for three months with a hydraulic retention time (HRT) of 32 h and a volumetric load of 0.28 kg COD m³ d^?1, the treatment process achieved mean removal efficiencies of 63.5% for chemical oxygen demand (COD), 45% for NH₃-N, 79.5% for total suspended solid (TSS), and 68.0% for total petroleum hydrocarbon (TPH). GC/MS was used to analyze relative changes of components of main organic waste in a process indicating that the influent wastewater contained organic compounds from C₁₂H₂₆ to C₃₅H₇₂, which could be degraded effectively with the coordinated action of hydrolysis acidification and aerobic treatment. The use of maize powder can enhance environmental adaptability of microorganisms and biodegradation ability and is recommended as a nutrient supplement to maintain good treatment performance.     

Impact of rock materials and biofertilizations on P and K availability for maize (Zea Maize) under calcareous soil conditions

The present work evaluated the synergistic effects of soil fertilization with rock P and K materials and co-inoculation with P and K-dissolving bacteria [PDB (Bacillus megaterium var. phosphaticum) and KDB (Bacillus mucilaginosus and B. subtilis)] on the improvement of P and K uptake, P and K availability and growth of maize plant grown under limited P and K soil conditions (calcareous soil). The experiment was establishment with eight treatments: without rock P and K materials or bacteria inoculation (control), rock P (RP), rock K (RK), RP + PDB, RK + KDB and R(P + K)+(P + K)DB. Under the same conditions of this study, co-inoculation of PDB and KDB in conjunction with direct application of rock P and K materials (R(P + K)) into the soil increased P and K availability and uptake, and the plant growth (shoot and root growth) of maize plants grown on P and K limited soils.     

Livestock paths on Namaqualand quartz fields: Will the endemic flora disappear?

Quartz fields are rare features that contribute significantly to vegetation diversity and endemism of South Africa's Succulent Karoo Biome. The Riethuis-Wallekraal quartz fields in the north-western Namaqualand area of South Africa contain 17 quartz field specialist species of which seven are endemic to this specific area. Hoof-action by livestock has formed paths of approximately 0.30 m on these quartz fields. It would be important to conservationists to understand whether direct (e.g. trampling) and indirect effects (e.g. burial of flora by sediment movement) associated with the livestock paths holds any threat to the dwarf succulent (< 0.05 m) and micro-chamaephytes (0.06–0.15 m) endemic to the quartz fields. We tested the hypotheses that the unique quartz field vegetation and biological soil crusts would be affected by loose soil particles transported downslope from the paths. The soil stability index, total vegetation cover, cover of specialized quartz field species and species diversity were lower on livestock paths but did not differ between upslope and downslope locations. Livestock paths also had lower cover and fewer quartz field specialist species. It is concluded that under conditions of intense and continuous grazing, livestock are likely to have an even stronger negative impact on the specialist quartz field flora.     

Morphological,ultrastructural and phylogenetic analyses of Myxobolus hilarii n. sp. (Myxozoa,Myxosporea), a renal parasite of farmed Brycon hilarii in Brazil

Myxobolus hilarii n. sp. was described, based on morphology, histology, ultrastructure and 18S rDNA sequencing, infecting the kidney of Brycon hilarii (Valenciennes 1850) (Characiformes: Bryconidae) taken from fish farms in the state of São Paulo, Brazil. Thirteen specimens of B. hilarii were examined and 100% had round, white plasmodia in the kidney. The mature myxospores were rounded, measuring 11.5 ± 0.8 (9.8–13.4) μm in length, 11.0 ± 0.7 (9.7–12.4) μm in width and 7.6 ± 1.0 (6.7–9.0) μm in thickness. Polar capsules were elongated and of equal size, with 6.5 ± 0.4 (6.0–7.2) μm in length and 4.0 ± 0.2 (3.6–5.3) μm in width and their polar filaments had 5 to 7 coils. Histological analysis revealed plasmodial development in the renal tubules, causing compression and deformation of adjacent tissues and destruction of renal tubule cells. Ultrastructural analysis showed direct contact between the plasmodial wall and the host tissue and asynchronous plasmodial development. The phylogenetic analysis of South American myxobolids, based on 18S rDNA sequencing, showed the myxosporeans grouping into two main clades. M. hilarii n. sp. appears as sister species of Myxobolus piraputangae.     

Microhabitat variations and seed bank-vegetation relationships in a desert wadi ecosystem

Due to extreme variability in patterns of rainfall, plant seed banks are an important component of desert habitats. Here I report on effects of standing vegetation and three different microhabitats (channel, bank and terrace) on the soil seed bank of a desert wadi ecosystem in the Eastern Desert of Egypt. A total of 450 soil samples at 45 stands were collected to represent the different wadi microhabitats. The germinable seed bank was estimated by controlled counts of seedling emergence. The floristic composition, functional properties and diversity of the soil seed bank, as well as its similarity with the standing vegetation varied among wadi microhabitats. Such variation could be attributed to differences in disturbance intensity among microhabitats (terrace < bank < channel) and variation of soil factors along the microtopographic gradient. Channel showed the highest species richness and size of soil seed bank, followed by bank and then terrace. Moreover the Shannon index of diversity of the seed bank and its similarity with standing vegetation were significantly greater in both channel and bank microhabitats than in terrace. At the level of plant functional groups, number of seeds of annuals was higher in both channel and bank than in terrace. Shrubs were more abundant in seed banks of channel compared to terrace. The size and species richness of seed bank were increased with the total plant cover, annual/perennial ratio and species richness of the standing vegetation.     

Variation of soil nutrients and particle size under different vegetation types in the Yellow River Delta

The Yellow River Delta wetland, located at the southern coast of Bohai Gulf, provides important ecosystem services such as flood control, water purification, biodiversity conservation, nutrient removal and carbon sequestration, shoreline stabilization, tourism attraction and wetland products maintains in the Yellow River Delta. This study assessed how agricultural activities in a reclamation wetland changed soil pH, soil electric conductivity, soil nutrient and soil particle size as compared to natural vegetation by using a combination of field experiments and lab analysis. The vegetation type included adjacent alfalfa field (Medicago sativa), cotton field (Gossypium spp.), Chinese tamarisk shrub (Tamarix chinensis), and reed marsh (Phragmites sage). The results indicated that the soil pH was higher (pH > 8) in alfalfa field and cotton field, and alfalfa field and reed marsh had significant function in reducing soil salt content, soil electric conductivity of alfalfa field at 0–30 cm were 140.38 ± 14.36, 114.48 ± 14.36, 125.30 ± 11.37 μs/cm. The effect of different vegetation types on soil nutrient was significant (P < 0.05). Soil organic matter at 0–10 cm in Chinese tamarisk shrub and reed marsh was 21.66 ± 3.82 g/kg and 16.51 ± 4.60 g/kg, which was higher than that of alfalfa field (10.47 ± 2.36 g/kg) and cotton field (9.82 ± 1.27 g/kg), but soil total nitrogen content in alfalfa field was the highest, which is significantly higher than that of cotton field, Chinese tamarisk shrub and reed marsh(P < 0.05), the content of soil total nitrogen at 0–10 cm and 10–20 cm was 7.67 ± 0.38 g/kg and 5.97 ± 0.51 g/kg, respectively, while the content of available P and available K was reversed. The difference of soil particle size between layers was not significant (P > 0.05), the sand content of Chinese tamarisk shrub soils in 0–10 cm was the highest, the next was alfalfa field and cotton field, and the content of silt and clay in reed marsh was higher than the others. The correlation and significant degree between soil particle size and soil nutrient was related with vegetation types, and soil organic matter was significantly positively correlated with soil silt and clay content on the alfalfa field. The results demonstrated that wetland cultivation was one of the most important factors influencing on the nutrient fate and reserves in soil, which could lead to rapid nutrient release and slow restoration through abandon cultivation. Consequently, compared with cotton field, alfalfa field is more favorable to sustainable management of wetland cultivation in the Yellow River Delta. It should be considered in wetland restoration projects planning.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 μg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 μg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 μg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 × 10² to 1 × 10⁵ genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 × 10⁵ CFU/ml at 4, 20, and 37 °C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 °C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 °C to a minority of the mixed population compared to membrane damage.     

Evaluation of hexavalent chromium reduction by chromate-resistant moderately halophile,Nesterenkonia sp. strain MF2

A Gram-positive moderately halophilic chromate reducing bacterial strain was isolated from effluents of tanneries, and identified as Nesterenkonia sp. strain MF2 by phenotypic characterization and 16S rRNA analysis. The strain could tolerate up to 600 mM of chromate and completely reduced 0.2 mM highly toxic and soluble Cr(VI) (as CrO₄²⁻) into almost non-toxic and insoluble Cr(III) in 24 h under aerobic condition.The maximum chromate removal was exhibited in 1.5 M NaCl at 35 °C and pH 8.0. Initial Cr(VI) concentration until 0.4 mM did not have a significant effect on Cr(VI) reduction. The isolate was capable of chromate reduction in the presence of various concentrations of salts. The chromate reduction corresponded with growth of bacteria and reached a maximum level at the end of exponential phase.     

Shrub removal facilitates recovery of wetland species in a rewetted fen

Drainage and shrub expansion are the main threats to the biodiversity of fens and fen meadows, whereas rewetting and the removal of shrubby species are frequently applied restoration measures. We examine whether removal of shrubs enhances recovery of target species in a degraded fen subjected to moderate rewetting. The study was located in the drained fen Ca?owanie (central Poland), where remnants of open fen communities and willow-invaded fens exist in former turf-pits, surrounded by degraded meadows on dried peat. All these three habitat types were included in a monitoring grid, which covered an area of 2.2 ha. Within 55 quadrats of 20 m × 20 m we monitored occurrence of 52 species, i.e. two groups of target species (fen indicators and wet meadow indicators) and indicators of two failure scenarios (degraded fen indicators and eutrophic wetland indicators), during six years following shrub removal, rewetting and re-application of conservational mowing, using a 3-step ordinal abundance scale. NMDS ordination revealed a gradual convergence of shrub removal plots and reference plots. We noticed significant effects of year and habitat type on all indicator groups, but only fen indicators have shown a clear (increasing) trend within shrub removal plots. Degraded fen indicators (ruderal and opportunistic species) initially expanded on shrub removal plots, but this effect disappeared in the following years. We conclude that shrub removal enhances establishment of target species in a moderately drained and then rewetted fen and attribute this effect to lowered competition for light. However, given high costs of this method and long-lasting problems with shrub resprouts, we recommend applying shrub removal only to recently overgrown sites, which still retained high botanical diversity. Heavily degraded fen meadows did not react on the increase of moisture, which indicates that more advanced restoration measures, such as top soil removal are needed there.     

Development of a microtitre plate method for determination of phenol utilization,biofilm formation and respiratory activity by environmental bacterial isolates

Twenty-five aerobic phenol-degrading bacteria, isolated from different environmental samples on phenol agar after several subcultures in phenol broth, utilized phenol (0.2 g l⁻¹) within 24 h, but removal of phenol was more rapid when other carbon sources were also present. A microtitre plate method was developed to determine growth rate, biofilm formation and respiratory activity of the strains isolated. Pseudomonas putida strains C5 and D6 showed maximum growth (as O.D. at 600 nm), P. putida D6 and unidentified bacterial strain M1 were more stable at high concentrations of phenol (0.8 g l⁻¹), and P. putida C5 formed the greatest amount of biofilm in 0.5 g phenol l⁻¹ medium. Measurement of dehydrogenase activity as reduction of triphenyl tetrazolium chloride supported data on growth rate and biofilm formation. The microtitre plate method provided a selective method for detection of the best phenol degrading and biofilm-forming microorganisms, and was also a rapid, convenient means of studying the effect of phenol concentration on growth rate and biofilm formation.