An LED-based UV-B irradiation system for tiny organisms: System description and demonstration experiment to determine the hatchability of eggs from four Tetranychus spider mite species from Okinawa

We developed a computer-based system for controlling the photoperiod and irradiance of UV-B and white light from a 5 × 5 light-emitting diode (LED) matrix (100 × 100 mm). In this system, the LED matrix was installed in each of four irradiation boxes and controlled by pulse-width modulators so that each box can independently emit UV-B and white light at irradiances of up to 1.5 and 4.0 W m⁻², respectively, or a combination of both light types. We used this system to examine the hatchabilities of the eggs of four Tetranychus spider mite species (T. urticae, T. kanzawai, T. piercei and T. okinawanus) collected from Okinawa Island under UV-B irradiation alone or simultaneous irradiation with white light for 12 h d⁻¹ at 25 °C. Although no eggs of any species hatched under the UV-B irradiation, even when the irradiance was as low as 0.02 W m⁻², the hatchabilities increased to >90% under simultaneous irradiation with 4.0 W m⁻² white light. At 0.06 W m⁻² UV-B, T. okinawanus eggs hatched (15% hatchability) under simultaneous irradiation with white light, whereas other species showed hatchabilities <1%. These results suggest that photolyases activated by white light may reduce UV-B–induced DNA damage in spider mite eggs and that the greater UV-B tolerance of T. okinawanus may explain its dominance on plants in seashore environments, which have a higher risk of exposure to reflected UV-B even on the undersurface of leaves. Our system will be useful for further examination of photophysiological responses of tiny organisms because of its ability to precisely control radiation conditions.     

Impact of soil mesofauna on the decomposition of two main species litters in a Pinus koraiensis mixed broad-leaved forest of the Changbai Mountains

In most terrestrial ecosystems, the majority of aboveground net primary productivity enters the decomposition system as plant litter. The decomposition of plant litter plays a critical role in regulating build up of the forest soil organic matter, releasing of nutrients for plant growth, and influencing the carbon cycling. Soil fauna are considered to be an important factor in the acceleration litter decomposition and nutrient transformations. Mechanisms of soil faunal contribution to litter decomposition include digestion of substrates, increase of surface area through fragmentation and acceleration of microbial inoculation into litter. The Pinus koraiensis mixed broad-leaved forest is one of the typical forest vegetation types in Changbai Mountain. Previously, major studies carried here were focused on climate, soil and vegetation; however, on litter decomposition and the role of soil fauna in this forest ecosystem were limited. In this paper, we conducted a litter decomposition experiment using litterbag method to explore the contribution of soil fauna on litter decomposition and provide a scientific basis for maintaining a balanced in P. koraiensis mixed broad-leaved forest in Changbai Mountains. During 2009 and 2010, we used litterbags with different mesh sizes to examine the decomposition of two dominant tree species (P. koraiensis, Fraxinus mandshurica) in studied site. The results showed that the process of litter decomposition can be separated into two apparent stages. The initial decomposition process at former six months was slow, while accelerated the final six months. The former six months (from October 2009 to April 2010) was winter and spring. There was low temperature and almost no activity of soil fauna and microbes. The final six months (from June to October 2010), decomposition rates increased. In summer and autumn, both temperature and moisture increases, abundance of soil fauna was much than before and was most active. The remaining mass of P. koraiensis was higher than that of F. mandshurica in two mesh size litterbags after 1 year decomposition, meanwhile litter in 2 mm mesh size litterbag had higher decomposition rate than that of 0.01 mm for two species litter. The Collembola, Acari, Enchytraeidae Lithobiomorpha and Diptera larvae were mainly fauna groups in the litterbags. The composition of soil fauna community was difference between P. koraiensis and F. mandshurica during litter decomposition. 24 different soil fauna groups and 1431 individual were obtained in P. koraiensis litterbags; Isotomidae, Tomoceridae and Oribatida were dominant groups; while 31 different soil fauna groups and 1255 individual were obtained in F. mandshurica litterbags; Isotomidae, Hypogastruridae Oribatida and Mesostigmata were dominant groups. The rate of litter decomposition was positively correlated with the individual and group density of soil fauna. Contribution rate to litter decomposition was 1.70% for P. koraiensis and 4.83% for F. mandshurica. Repeated measures ANOVA showed that litter species, time and soil fauna had a significant impact on the rate of litter decomposition (P < 0.05). Our results suggested that soil fauna could accelerate litter decomposition and, consequently, nutrient cycling in P. koraiensis mixed broad-leaved forest, Changbai Mountains.     

Does UV-B influence biomass growth in lichens deficient in sun-screening pigments?

This study aimed to assess biomass growth as a response variable in lichens during short-term laboratory experiments. To do this, we studied the influence of UV-B and temperature on lichen performance including the synthesis of solar radiation screening cortical compounds. The pioneer lichen Xanthoria aureola from exposed sea cliffs and the old forest lichen Lobaria pulmonaria were cultivated for 15 days in the laboratory in a factorial experiments with temperature (12 and 21 °C) and UV-B (0, 0.1, 0.3 and 1.0 W m^?2) as treatments. Prior to the experiment, the cortical pigment parietin was non-destructively extracted from X. aureola, whereas the sampled shade-adapted thalli of L. pulmonaria lacked cortical melanic compounds. Therefore both lichens were deficient in cortical sun-screening compounds when the UV-B exposure started. At 12 °C, the relative growth rate was 7.2 ± 0.6 and 3.0 ± 0.8 mg g^?1 day^?1 in L. pulmonaria and X. aureola, respectively, reduced to 1.8 ± 0.5 and ?2.6 ± 0.9 mg g^?1 day^?1, at 21 °C. These figures showed that lichen growth is a useful response variable in short-term laboratory experiments. Growth was not influenced by UV-B alone in these pigment-deficient transplants, suggesting that UV-B had little adverse effects on either of the lichen bionts. The cortical sun screens (parietin and melanic compounds) were synthesized in the presence of UV-B, and increased statistically significantly with increasing UV-B at both cultivation temperatures. However, in X. aureola the synthesis was highest at the lowest temperature (12 °C). At 12 °C, changes in chlorophylls, F_v/F_m and NPQ during cultivation were consistent with a substantial level of acclimation to the growth chamber conditions for both species, whereas strong reductions in photosynthetic pigments, F_v/F_m and Ф_II at 21 °C indicated serious damage and chlorophyll degradation at high temperature. In conclusion, lichen growth and the synthesis of protective compounds are highly responsive lichen processes in short-term experiments.     

The impacts of UV-B radiation on the regeneration of a sub-arctic heath community

The effects of ultraviolet-B radiation on regeneration after disturbance of a natural sub-arctic heathland have been investigated. Areas of pristine dwarf shrub heath were denuded of all above ground biomass in 1992 and exposed to enhanced UV-B (simulating a 15% depletion of the ozone layer). The resulting regenerated stem and leaf growth parameters were measured after four years on three dwarf shrubs, Vaccinium myrtillus, V. uliginosum and V. vitis-idaea and the grass Calamagrostis lapponica; leaves of the three dwarf shrubs were also analysed for UV-absorbing compounds and carbohydrates. Regeneration irrespective of treatment was slow, with Empetrum hermaphroditum failing to regenerate at all. Vaccinium myrtillus showed the most rapid regeneration attaining much of its original biomass in four years. There was a significant interaction between UV-B and year of regeneration in V. myrtillus; annual stem length increment showed an initial stimulation of 75% under enhanced UV-B in the first year of regeneration while a reduction of 16% was observed in the fourth year. Both V. uliginosum and V. vitis-idaea showed a reduction in annual stem length increment as regeneration progressed with a greater than 50% reduction in stem increment in the fourth year of regeneration compared to the first. Vaccinium uliginosum also showed an initial reduction in stem length increment of 40% under enhanced UV-B. None of the species were affected by enhanced UV-B in terms of total regenerated stem and leaf biomass or UV-absorbing compounds in regenerated leaf tissue. Total leaf carbohydrate and the ethanol/water soluble fraction in V. uliginosum were significantly increased by 29% and 31% respectively under enhanced UV-B. This suggests either a stimulation of photosynthesis or a reduction in sink size for photo-assimilates. Results are discussed in the context of the extremely slow regeneration of sub-arctic heath communities and the implications of contrasting UV-B effects on the regenerative ability of different species.     

Partial protection of photosynthetic apparatus from UV-B-induced damage by UV-A radiation

Alteration in the photosynthetic apparatus of clusterbean (Cyamopsis tetraganoloba) cotyledons owing to UV-B irradiation in the absence or presence of UV-A radiation (UV-A + UV-B) during steady phase of its growth has been studied. UV-B radiation induces a decline in the photosynthetic pigments content and O₂ evolution along with a modification in the absorption spectra of chloroplasts. UV-A + UV-B irradiation moderately reverses these changes. The partial restoration of F_V/F_M value and other fluorescence transient parameters in UV-A + UV-B treated sample compared to that of UV-B treated one suggest that UV-A helps in developing a protective pathway against UV-B-induced impairment. UV-B-mediated alteration in S state transition of Mn cluster associated with oxygen evolving complex, as appeared from TL glow curves, is retrieved by UV-A radiation and Car is considered to negotiate against UV-B-induced damage of photosynthetic apparatus.     

AhpC (alkyl hydroperoxide reductase) from Anabaena sp. PCC 7120 protects Escherichia coli from multiple abiotic stresses

Alkyl hydroperoxide reductase (AhpC) is known to detoxify peroxides and reactive sulfur species (RSS). However, the relationship between its expression and combating of abiotic stresses is still not clear. To investigate this relationship, the genes encoding the alkyl hydroperoxide reductase (ahpC) from Anabaena sp. PCC 7120 were introduced into E. coli using pGEX-5X-2 vector and their possible functions against heat, salt, carbofuron, cadmium, copper and UV-B were analyzed. The transformed E. coli cells registered significantly increase in growth than the control cells under temperature (47 °C), NaCl (6% w/v), carbofuron (0.025 mg ml^?1), CdCl₂ (4 mM), CuCl₂ (1 mM), and UV-B (10 min) exposure. Enhanced expression of ahpC gene as measured by semi-quantitative RT-PCR under aforementioned stresses at different time points demonstrated its role in offering tolerance against multiple abiotic stresses.     

Metabolism of terpenes in the response of grape (Vitis vinifera L.) leaf tissues to UV-B radiation

This study investigated the terpene profiles as determined by GC–EIMS analysis of in vitro cultured plants of Vitis vinifera exposed to a “field-like” dose of UV-B (4.75 kJ m⁻² d⁻¹) administered at two different fluence rates (low, 16 h at 8.25 μW cm⁻², and high 4 h at 33 μW cm⁻²). Low UV-B treatment increased levels of the membrane-related triterpenes sitosterol, stigmasterol and lupeol, more notable in young leaves, suggesting elicitation of a mechanism for grapevine acclimation. By contrast, accumulation of compounds with antioxidant properties, diterpenes α and γ tocopherol and phytol, the sesquiterpene E-nerolidol and the monoterpenes carene, α-pinene and terpinolene had maximum accumulation under high UV-B, which was accentuated in mature leaves. Also the levels of the sesquiterpenic stress-related hormone abscisic acid (ABA) increased under high UV-B, although 24 h post irradiation ABA concentrations decreased. Such increments of antioxidant terpenes along with ABA suggest elicitation of mechanism of defense. The adaptative responses induced by relatively low UV-B irradiations as suggested by synthesis of terpenes related with membrane stability correlated with augments in terpene synthase activity.     

Degradation of phenol by a halotolerant strain of Penicillium chrysogenum

The lowest 50% lethal (effective) concentration, L(E)C₅₀, of phenol in a battery of seven microbiotests with species representing different trophic levels was 1–10 mg l⁻¹, classifying it as “toxic”. A phenol-degrading microorganism was isolated from soil samples of the salt mine of Clona in Portugal, after enrichment in the presence of phenol and high salt concentration. Based on cultural and morphological characteristics, the strain CLONA2 was identified as belonging to Penicillium chrysogenum. It was found to be a halotolerant fungus able to grow in a nutrient-rich medium with 5.8% NaCl. It degraded at least 300 mg l⁻¹ phenol as sole source of carbon and energy, without accumulation of intermediates. The samples were also tested for toxicity using the Microtox^® assay. Data showed that P. chrysogenum CLONA2 could be effectively utilized to reduce phenol toxicity. The results suggest also that phenol under saline conditions can be successfully mineralized by P. chrysogenum CLONA2.     

Studying the cytotoxicity and oxidative stress induced by two kinds of bentonite particles on human B lymphoblast cells in vitro

The aim of the present study was to evaluate the cytotoxicity and oxidative stress induced by native and active bentonite particles (BPs) on human B lymphoblast cells using seven assays. Our results showed that the order of cytotoxicity was: active BPs > native BPs > quartz particles (DQ-12) > gypsum, according to the IC50 values in CCK-8 assay and neutral red uptake (NRU) assay. The lactate dehydrogenase (LDH) leakage, the proportions of early apoptotic cells, the reactive oxygen species (ROS) generation, the superoxide dismutase (SOD) inhibition and the malondialdehyde (MDA) release in the native and active BPs groups were significantly higher than those in the gypsum and DQ-12 groups (P < 0.05 or P < 0.01). Moreover, the cytotoxicity of active BPs with higher adsorption capacity of phenol was higher than that of native BPs with relatively lower adsorption capacity of phenol. The oxidative stress induced by active BPs was significantly higher than that induced by native BPs (P < 0.05 or P < 0.01). The water-soluble fractions of BPs did not induce the cytotoxicity and ROS generation. These findings indicated that active and native BPs could induce significantly the cytotoxic effects and oxidative stress on human B lymphoblast cells in vitro. The cytotoxic difference between active BPs and native BPs may be associated with the adsorption capacity of BPs and oxidative stress induced by BPs to a certain extent. The insoluble particle fractions may play a main role in the cytotoxic effects and oxidative stress induced by BPs.     

Exposures to elevated CO2, elevated temperature and enhanced UV-B radiation modify activities of polyphenol oxidase and guaiacol peroxidase and concentrations of chlorophylls,polyamines and soluble proteins in the leaves of Betula pendula seedlings

The activity of polyphenol oxidase (PPO) and guaiacol peroxidase (POD) and the concentrations of chlorophylls, free polyamines and soluble proteins were determined from the leaves of six genotypes of silver birch (Betula pendula Roth) seedlings exposed to short-term elevated carbon dioxide (CO₂), temperature (T), ultraviolet-B irradiation (UV-B, 280-315 nm) and their combinations. Results showed that the activity of PPO in the leaves was low but increased by elevated CO₂ and elevated T. The POD activity varied between the genotypes due to an interactive effect of CO₂ × UV-B. The soluble proteins were clearly decreased by elevated CO₂, but the level of response varied among the genotypes. The concentrations of chl a and total chlorophylls were lower in the leaves treated with elevated CO₂ than in leaves grown at ambient CO₂. An interactive effect of CO₂ × UV-B on the chl a/b ratio was found. Elevated T increased chl b concentration and decreased chl a/b ratio. Temperature treatments also caused variation in the concentrations of chl a, chl b and total chlorophylls among the genotypes. Polyamine analyses showed that the concentrations of putrescine were increased and spermine decreased in leaves treated with elevated T. However, the change in putrescine by elevated T was clearer at ambient CO₂ than in eCO₂ environment (significant effect of T × CO₂). In conclusion, the defensive enzymes, photosynthetic pigments, soluble proteins and growth-regulating polyamines in silver birch leaves were not susceptible to enhanced UV-B radiation. In contrast, all the variables responded to elevated T and/or elevated CO₂, reflecting the enhancive effects of climate change conditions not only on leaf productivity, but also on leaf turn-over rate. Most of these climate-driven changes were not regulated by UV-B radiation.     

Effects of nitrogen and phosphorus availability on the decomposition of aquatic plants

The responses of decomposition to nitrogen (N) and phosphorus (P) supply were investigated in three leaf species: Eichhornia crassipes, Vallisneria natans, and Potamogeton maackianus. Decomposition was fastest in E. crassipes (0.047–0.099 day⁻¹), intermediate in V. natans (about 0.030 day⁻¹), and slowest in P. maackianus (about 0.010 day⁻¹). Increase in P-availability increased the decomposition rate of E. crassipes by 68–87%, whereas the impact of N-availability alone was insignificant. Both N- and P-availability in waters had no significant impact on the decomposition rates of V. natans and P. maackianus (P > 0.05). The effects of P-availability on the N and P content levels of the three species were significant (P < 0.01), except for the impact on N content of V. natans (P = 0.526). In contrast, environment N-availability was insignificant. These results indicate that the responses of decomposition to nutrient availability depend on plant species and nutrient type. P-availability has stronger effects on litter nutrient dynamics than N-availability.     

Effects of experimental warming and nitrogen enrichment on leaf and litter chemistry of a wetland grass,Phragmites australis

As global climate is warming and the nitrogen cycle accelerates, plants are likely to respond not only by shifting community composition, but also by adjusting traits such as tissue chemistry. We subjected a widespread wetland plant, Phragmites australis, to increased nitrate supply and elevated temperature in enclosures that were established in a littoral permanently submerged freshwater marsh. The nitrogen (N) and phosphorus (P) concentrations in green leaves ranged from 11.4 to 13.8 mg N and from 1.5 to 2.0 mg P g⁻¹ dry mass. While the N concentration changed little in brown litter, the P concentration decreased to 0.53–0.65 mg P g⁻¹ litter dry mass. Neither experimental warming of the water and sediment surface, nor nitrate enrichment during the growing season affected nitrogen or phosphorus concentrations in green leaves. Concentrations of the two major structural carbon compounds in plant litter, cellulose and lignin, were also unaffected, ranging from 32.1 to 34.2% of dry mass for cellulose and from 16.3 to 17.7% of dry mass for lignin. Warming, however, significantly increased the nitrogen concentration of fully brown leaf litter. Thus, temperature appears to be more important than the supply of dissolved N in the water, especially in affecting leaf litter N concentrations in P. australis, even when only water but not air temperature is increased. This result may have implications for decomposition processes and decomposer food webs, which both depend on the quality of plant litter.     

Development of permethrin resistance in Culex quinquefasciatus Say in Kuala Lumpur,Malaysia

The resistance status towards permethrin among the laboratory strain, the permethrin-selected strain and four field strains of Culex quinquefasciatus collected in Kuala Lumpur, Malaysia was determined using three standard laboratory methods: WHO larval bioassay, WHO adult bioassay and biochemical microplate assay. Cx. quinquefasciatus permethrin-selected strain larvae were the least susceptible to permethrin with a resistance ratio of 47.28-folds, whereas all field strain larvae of the same species were tolerant to permethrin with resistance ratios of more than 3-folds. In contrast, in adult stage, the permethrin exposed permethrin-selected strain (resistance ratio = 1.27) was found to be more susceptible to permethrin than all permethrin-exposed field strains (resistance ratios = 2.23–2.48). Complete mortalities for all strains of Cx. quinquefasciatus adults proved the effectiveness of the synergist; piperonyl butoxide (PBO). For the biochemical microplate assay, the reduction of the mean optical density of elevated oxidase activity of three field strains upon exposure to PBO confirmed the association between oxidase activity and permethrin tolerance. On the other hand, irregular patterns of the mean optical density of elevated oxidase activity in the laboratory strain, permethrin-selected strain and Jalan Fletcher strain illustrated the gene variation within these mosquito colonies as well as the involvement of other enzyme activities in the permethrin resistance occurred.     

Chara hispida beds as a sink of nitrogen: Evidence from growth,nitrogen uptake and decomposition

Chara hispida forms dense beds (0.78–0.95 kg DW m⁻²) in Colgada Lake. The ability of Chara meadows to act as a nitrogen source or sink was evaluated by the following methods: (1) investigating Chara growth, (2) nitrogen incorporation and decomposition laboratory experiments and (3) relating experimental results to field conditions. Sediment oospores were germinated in large aquaria and observed growth rates were 0.001 m day⁻¹ (shoot length) and 0.0002 g day⁻¹ (dry weight). Nitrogen uptake rates were determined by addition of K¹⁵NO₃ during two different periods of Chara growth and the rates were 1.21 and 3.86 μM g DW⁻¹ h⁻¹ when charophytes were 166 days old (not sexually mature) and 323 days old (sexually mature), respectively. After the uptake experiments, the same charophytes were allowed to decompose within two types of litter bags (3 mm-pore litter bags and entire, non-porous plastic litter bags). Decomposition rates of Ch. hispida were 0.016 and 0.009 day⁻¹ in perforated and non-perforated bags, respectively, and fit a negative exponential model. The nitrogen release rate, calculated as the disappearance of N content from Chara tissues, was 0.012 day⁻¹ and there were no statistically significant differences between the values from the two different bag types. The dissolved organic nitrogen concentrations in aquarium and non-perforated litter bags waters increased linearly with time due to the leaching of soluble compounds from Chara. The rate of N loss from Chara tissues, total nitrogen and dissolved organic nitrogen release rates and the decrease in initial dry weight rate were all lower than the daily rate of Chara N uptake. By extrapolating laboratory data to field situations, we determined that approximately 38% of the N taken up by charophytes in Colgada Lake during the growth period is retained. Given the high charophyte biomass in the lake, its ability to incorporate nitrogen, its low decomposition rate and its ability to over-winter, we conclude that Chara beds could be acting as nitrogen sinks in this ecosystem.     

Effects of low UV-B doses on the accumulation of UV-B absorbing compounds and total phenolics and carbohydrate metabolism in the peel of harvested lemons

This paper examined the peel (albedo and flavedo) of postharvest lemon fruits after UV-B exposure in order to analyze relationships between soluble carbohydrate metabolism and secondary metabolite accumulation. Lemons (Citrus limon, cv. Limoneira 8A) were harvested in winter months (June to August), treated with 0.43 W m⁻² (22 kJ m⁻² d⁻¹ UV-BBE) of UV-B radiation during 0 (control), 0.5, 1.0, 2.0, 3.0, and 5.0 min, and then stored at 25 °C for 24 h. Peel samples from irradiated areas were obtained with a razor blade and frozen in liquid nitrogen until use for measurements. Data obtained showed that 2 and 3 min of UV-B exposure effectively increased the level of UV-B absorbing compounds and total phenolics in flavedo without causing visual alterations of the peel colour as compared with non-irradiated lemons. By contrast, there were no significant changes in albedo secondary metabolite accumulation. The amount of secondary metabolites was depending upon UV-B time–dose. Exposure over 3.0 min did not further improve the accumulation of UV-B absorbing and phenolic compounds. Soluble sugars (sucrose, glucose and fructose) also accumulated in the lemon peel after UV-B exposure, but the distribution patterns were different. After 3 min time–dose, sucrose and hexoses increased in flavedo, whereas in albedo only increased the sucrose and glucose. This effect was related to UVB-induced changes in the activity of sucrose-hydrolyzing and sucrose-synthesizing enzymes: soluble and cell-bound invertase, sucrose synthase (SS) and sucrose phosphate synthase (SPS). Data indicate that lemon peel retains the capacity to modify the enzyme activity of sucrose metabolism in response to UV-B exposure. Our results also suggest that the exposure of postharvest lemons to low supplemental UV-B doses produces changes in the carbon allocation of peel tissues including synthesis, but probably not only limited to them, of UV-B absorbing and phenolic compounds.     

Enrichment,isolation, and characterization of phenol-degrading Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6

The objectives of this research were to isolate pure phenol-degrading strains from enriched mixed cultures, monitoring the variations of species during the enrichment period. Two strains were isolated from the acclimated mixed culture. They were identified as Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6. DGGE indicated that strain P. resinovorans appeared at the beginning, and maintained well during the enrichment period. The second strain, Brevibacillus sp., did not appear in the initial stage, but showed up after 2 weeks of enrichment. The optimum growth temperatures for P. resinovorans and Brevibacillus sp. were 31 and 39 °C, respectively. P. resinovorans could degrade phenol completely within 57.5 h, when the initial phenol concentration was lower than 600 mg l⁻¹. If the initial phenol concentration was lower than 200 mg l⁻¹, Brevibacillus sp. could remove phenol completely within 93.1 h. It was obvious that the phenol-degrading ability of P. resinovorans was much better than that of Brevibacillus sp. The metabolic pathway for P. resinovorans phenol degradation was assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.     

Isolation and characterization of ellagitannins as the major polyphenolic components of Longan (Dimocarpus longan Lour) seeds

Longan (Dimocarpus longan Lour, syn. Euphoria longan Lam.) represents an important fruit in Northern Thailand and has significant economic impact. The fruit is either consumed fresh or as commercially prepared dried and canned products. The canning industry in Thailand produces considerable quantities of waste products, in particular Longan seeds. Because these seeds may be an exploitable source of natural phenolic antioxidants, it was of interest to identify, purify and quantitate the major potential antioxidant phenolics contained therein. The polyphenolic fraction from ground Longan seeds was obtained by extraction with methanol after delipidation with hexane. The hexane extract contained predominantly long-chain fatty acids with major contributions from palmitic (35%) and oleic (28%) acids. The polyphenolic fraction (80.90 g/kg dry weight) was dominated by ellagic acid (25.84 g/kg) and the known ellagitannins corilagin (13.31 g/kg), chebulagic acid (13.06 g/kg), ellagic acid 4-O-α-l-arabinofuranoside (9.93 g/kg), isomallotinic acid (8.56 g/kg) and geraniin (5.79 g/kg). Structure elucidation was performed with mass spectrometry and complete assignment of ¹H and ¹³C NMR signals. The methanol extracts exhibited strong antioxidant capacities with an IC₅₀ of 154 μg/ml for reactive oxygen species attack on salicylic acid and 78 μg/ml for inhibition of xanthine oxidase in the hypoxanthine/xanthine oxidase assay. The extracts were less effective in the 2-deoxyguanosine assay (IC₅₀ = 2.46 mg/ml), indicating that gallates along with ellagic acid and its congeners exert their potential antioxidant effects predominantly by precipitation of proteins such as xanthine oxidase. This was confirmed for the pure compounds gallic acid, methyl gallate, ellagic acid and corilagin.     

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.     

Phenol and sulfide oxidation in a denitrifying biofilm reactor and its microbial community analysis

A microbial consortium attached onto a polyethylene support was used to evaluate the simultaneous oxidation of sulfide and phenol by denitrification. The phenol, sulfide and nitrate loading rates applied to an inverse fluidized bed reactor were up to 168 mg phenol–C/(l d), 37 mg S^2?/(l d) and 168 mg NO₃^?–N/(l d), respectively. Under steady state operation the consumption efficiencies of phenol, sulfide and nitrate were 100%. The N₂ yield (g N₂/g NO₃^?–N) was 0.89. The phenol was mineralized resulting in a yield of 0.82 g bicarbonate–C/g phenol–C and sulfide was completely oxidized to sulfate with a yield of 0.99 g SO₄^2?–S/g S^2?. 16S rRNA gene-based microbial community analysis of the denitrifying biofilm showed the presence of Thauera aromatica, Thiobacillus denitrificans, Thiobacillus sajanensis and Thiobacillus sp. This is the first work reporting the simultaneous oxidation of sulfide and phenol in a denitrifying biofilm reactor.     

Vibrio aestivus sp. nov. and Vibrio quintilis sp. nov., related to Marisflavi and Gazogenes clades,respectively

Two new Vibrio species, Vibrio aestivus and Vibrio quintilis, are described after a polyphasic characterization of strains M22^T, M61 and M62^T, isolated from seawater collected off a beach on the East coast of Spain (Valencia). All three strains are Gram negative, mesophilic, slightly halophilic, fermentative rods. V. aestivus (M22^T = CECT 7558^T = CAIM 1861^T = KCTC 23860^T and M61 = CECT 7559 = CAIM 1862 = KCTC 23861) is oxidase positive, reduces nitrates to nitrites, is negative for Voges Proskauer, arginine dihydrolase and indole and non hydrolytic on most substrates tested. The 16S rRNA gene sequences of M22^T and M61 are most similar to Vibrio marisflavi (97.1–97.2%) but phylogenetic analysis using NJ, MP and ML methods display Vibrio stylophorae (96.2% similarity) as sibling species. The three species form a deep clade in the genus Vibrio. Average Nucleotide Identity (ANI) values, determined as a measure of overall genomic resemblance, confirmed that strains M22^T and M61 are members of the same species, different to V. marisflavi CECT 7928^T.V. quintilis (M62^T = CECT 7734^T = CAIM 1863^T = KCTC 23833^T) is aerogenic, arginine dihydrolase and Voges Proskauer positive, oxidase negative and unable to reduce nitrate, traits shared by most species in the Gazogenes clade. It is unpigmented and does not grow on TCBS Agar. 16S rRNA gene similarities to its nearest species, Vibrio aerogenes and Vibrio mangrovi, are 97.6% and 96.0% respectively. Strain M62^T and V. aerogenes CECT 7868^T display ANI values well below the 95% boundary for genomic species.