Can vertical-flow wetland systems treat high concentrated sludge from a food industry? A mesocosm experiment testing three plant species

We performed a mesocosms experiment using a vertical-flow wetland system to treat liquid sludge in the Mediterranean region. Three common helophyte species, common reed (Phragmites australis Cav.), broadleaf cattail (Typha latifolia L.), and yellow flag (Iris pseudacorus L.), were planted as monoculture and irrigated with a liquid sewage sludge from a food industry, characterised by very high organic concentrations (COD > 8000 mg/L). We studied the benefits of plants by comparing unplanted to planted mesocosms. Results showed the high performance of such vertical-flow wetland systems. Removal efficiency was more than 98% for total suspended solids (TSS) and chemical oxygen demand (COD), and more than 87% for total Kjeldahl nitrogen (TKN). The main removal process was physical filtration by the substrate due to the high proportion of particulate elements in the sludge. Planted mesocosms were more efficient than those unplanted, confirming the positive role of the plants. Mesocosms planted with Phragmites or Typha showed better performances in TKN removal than those planted with Iris. Only in mesocosms planted with Phragmites was there no outflow in summer due to high evapotranspiration.     

Effect of hydraulic retention time on the treatment of secondary effluent in a subsurface flow constructed wetland

This study evaluates the potential of subsurface flow (SSF) constructed wetlands (CWs) for tertiary treatment of wastewater at four shorter HRTs (1–4 days). The CWs were planted with Typha angustata, which was observed in our earlier study to be more efficient than Phragmites karka and Scirpus littoralis. The CWs comprised four rectangular treatment cells (2.14 m × 0.76 m × 0.61 m) filled with layers of gravel of two different sizes (approximately 2.5 cm and 1.5 cm diameter) to a depth of 0.61 m. The inflow rates of the secondary effluent in the four cells were accordingly fixed at 300 L d^?1, 150 L d^?1, 100 L d^?1 and 75 L d^?1, respectively, for 1, 2, 3 and 4 days HRT. The hydraulic loads ranged between 59.05 mm d^?1 and 236.22 mm d^?1.The wastewater inflow into the CW system as well as the treated effluent were analyzed, using standard methods, at regular intervals for various forms of nitrogen (NH₄-N, NO₃-N and TKN), orthophosphate-P and organic matter (BOD and COD) concentrations over a period of five weeks after the development of a dense stand.The higher HRT of 4 days not only helped maximum removal of all the pollutants but also maintained the stability of the treatment efficiency throughout the monitoring period. For the nutrients (NH₄-N, NO₃-N and TKN), HRT played a more significant role in their removal than in case of organic matter (BOD₃ and COD). More than 90% of NO₃-N and TKN and 100% of NH₄-N were removed from the wastewater at 4 days HRT.At lower HRTs, the mass loading rate was higher with greater fluctuation. However mass reduction efficiency of the T. angustata CW for all forms of nitrogen was >80% with the HRTs of 2, 3 and 4 days.     

Comparison of lipopeptide biosurfactants production by Bacillus subtilis strains in submerged and solid state fermentation systems using a cheap carbon source: Some industrial applications of biosurfactants

Biosurfactants, in general has the potential to aid in the recovery of subsurface organic contaminants (environmental remediation) or crude oils (oil recovery). However, high production and purification costs limit its use in these high-volume applications. In the present study, the efficiency of two Bacillus subtilis strains viz., DM-03 and DM-04 for the production of biosurfactants in two fermentation systems viz., solid state fermentation (SSF) and submerged fermentation (SmF) was compared. Both the B. subtilis strains produced appreciable and equal amount of crude lipopeptide biosurfactants (B. subtilis DM-03: 80.0 ± 9 mg/gds in SmF and 67.0 ± 6 mg/gds in SSF; B. subtilis DM-04: 23.0 ± 5.0 mg/gds in SmF and 20.0 ± 2.5 mg/gds in SSF) in the two different fermentation systems using potato peels as cheap carbon source. These thermostable lipopeptide biosurfactants produced by B. subtilis strains either in SSF or in SmF, exhibited strong emulsifying property and could release appreciable amount of oil from saturated sand pack column. Further, it was shown by biochemical analysis, RP-HPLC profile and IR spectra that there is no qualitative and qualitative differences in the composition of crude biosurfactants produced either in SmF or in SSF system.     

Factors influencing the return of submerged plants to a clear-water,shallow temperate lake

Lack of submerged vegetation was studied in a small, shallow, alkaline, clear-water lake with high nitrate concentration (mean 9 mg NO₃–N L⁻¹) and profuse filamentous green algae (FGA) (mainly Spirogyra sp.). A laboratory microcosm and two lake enclosure experiments were carried out using Elodea nuttallii (Planchon) St John. E. nuttallii grew about 1.7 times as well in sediment from its place of origin compared with sediment from the lake. Differential water quality had no effect, and neither sediment nor water prevented growth in the lake. Nutrient addition reduced plant growth by more than 55% because of shading from epiphytic filamentous green algae (shoot dry weight versus epiphytic algal dry weight, r = −0.491, P < 0.05). Transplanted Elodea plants grew better in enclosures in the lake than in laboratory conditions with lake water and sediment (P < 0.001, t-test). Rare Elodea individuals in the lake indicate the presence of plant propagules in the lake sediment, but excessive growth of filamentous green algae (summer mean 3.2 g dry weight m⁻²) significantly hamperd plant growth (shoot length reduced from 29 ± S.E.M. 1 to 25 ± 1 cm) and bird herbivory significantly reduced survival (from 82 ± 7 to 40 ± 6%) and shoot growth (from 78 ± 6 to 18 ± 5 cm) and thus eliminates establishment of even modest plant beds. Fish disturbance and sediment stability were not important. Restoration of submerged plants may require reduction of nitrate input, control of filamentous green algae and protection from birds.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

Kinetics of pollutant removal from domestic wastewater in a tropical horizontal subsurface flow constructed wetland system: Effects of hydraulic loading rate

The treatment capacity of constructed wetlands is expected to be high in tropical areas because of the warm temperatures and the associated higher rates of microbial activity. A pilot scale horizontal subsurface flow constructed wetland system filled with river sand and planted with Phragmites vallatoria (L.) Veldkamp was set up in the southern part of Vietnam to assess the treatment capacity and the removal rate kinetics under tropical conditions. The system received municipal wastewater at four hydraulic loading rates (HLRs) of 31, 62, 104 and 146 mm day^?1. Removals of TSS, BOD₅ and COD were efficient at all HLRs with mean removal rates of 86–95%, 65–83% and 57–84%, respectively. Removals of N and P decreased with HLRs and were: NH₄-N 0–91%; TN 16–84% and TP 72–99%. First-order area-based removal rate constants (k, m year^?1) estimated from sampling along the length of the wetland from inlet to outlet at the four HLRs were in the range of 25–95 (BOD₅), 22–30 (COD), 31–115 (TSS), 5–24 (TN and TKN) and 41–84 (TP) at background concentrations (C*) of 5, 10, 0, 1.5 and 0 mg L^?1, respectively. The estimated k-values should not be used for design purposes, as site-specific differences and stochastic variability can be high. However, the study shows that domestic wastewater can be treated in horizontal subsurface flow constructed wetland systems to meet even the most stringent Vietnamese standards for discharge into surface waters.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Effects of intraspecific competition on growth and photosynthesis of Atriplex prostrata

We investigated the effect of intraspecific competition on growth parameters and photosynthesis of the salt marsh species Atriplex prostrata Boucher in order to distinguish the effects of density-dependent growth inhibition from salt stress. High plant density caused a reduction of 30% in height, 82% in stem dry mass, 80% in leaf dry mass, and 95% in root dry mass. High density also induced a pronounced 72% reduction in leaf area, 29% decrease in length of mature internodes and 50% decline in net photosynthetic rate. The alteration of net photosynthesis paralleled growth inhibition, decreasing from 7.6 ± 0.9 μmol CO₂ m⁻² s⁻¹ at low density to 3.5 ± 0.4 μmol CO₂ m⁻² s⁻¹ at high density, indicating growth inhibition caused by intraspecific competition is mainly due to a decline in net photosynthesis rate. Plants grown at high density also exhibited a reduction in stomatal conductance from 0.7 ± 0.1 mol H₂O m⁻² s⁻¹ at low density to 0.3 ± 0.1 mol H₂O m⁻² s⁻¹ at high density and a reduction in transpiration rate from 6.0 ± 0.3 mmol H₂O m⁻² s⁻¹ at low density to 4.3 ± 0.3 mmol H₂O m⁻² s⁻¹ at high density. Biomass production was inhibited by an increase in plant density, which reduced the rate of photosynthesis, stomatal conductance and leaf area of plants.     

Cyanobacterial biomass as N-supplement to agro-waste for hyper-production of laccase from Pleurotus ostreatus in solid state fermentation

The potential application of dry biomass of a cyanobacterium Anacystis nidulans as a supplement in SSF for the production of laccase from Pleurotus ostreatus was evaluated. Experiments were carried out in solid culture using groundnut shell as a basic substrate supplemented with four independent nitrogen sources (ammonium sulphate, urea, yeast extract and dry powder of cyanobacteria). All the four supplements enhanced the enzyme yield, and yeast extract showed precedence over inorganic nitrogenous sources. However, when dry biomass of A. nidulans was used as an additive to groundnut shell (agricultural residues), it supported maximum cell growth (56.83 ± 5.56 mg/g dry substrate) and laccase production (49.21 ± 4.89 U/g dry substrate). Addition of 1 mM copper salt in the medium containing groundnut shell supplemented with yeast extract gave laccase activity of 32.64 ± 3.4 U/g dry substrate. When dry powder of cyanobacterial biomass was used as N-supplement, laccase production enhanced to 65.42 ± 6.48 U/g dry substrate. In addition to the enhancement to enzyme production inhibitory effects of high concentrations of copper was also diminished in the medium having dry cyanobacterial biomass. This study, forms the first report on the potential application of cyanobacterial biomass as an additive for production of laccase by Pleurotus ostraetus MTCC 1804 in solid state fermentation and has relevance in scale-up production of this fungal enzyme of commercial significance.     

Treatment of domestic wastewater in tropical,subsurface flow constructed wetlands planted with Canna and Heliconia

Constructed wetlands have a good potential for wastewater treatment in developing countries due to the simple operation and low implementation costs. Ornamental plants like Canna and Heliconia are used in the wetlands to increase their aesthetic value and these two species were compared in this study. Six pilot scale horizontal subsurface flow constructed wetland units were constructed at the Asian Institute of Technology (AIT) campus in Bangkok, Thailand, of which three were planted with Heliconia psittacorum L.f. × H. Spathocircinata (Aristeguieta) and three with Canna × generalis L. Bailey. The beds were loaded with domestic wastewater in four trials with hydraulic loading rates ranging from 55 to 440 mm d^?1 corresponding to nominal detention times between 12 h and 4 days. Both plant species grew well in the systems and especially Canna had high growth rates (3100 ± 470 g DW m^?2 yr^?1) compared to Heliconia (550 ± 90 g DW m^?2 yr^?1). TSS mass removal rates were very high with efficiencies >88% even at hydraulic loading rates of 440 mm d^?1. COD mass removal rates varied between 42 and 83% depending on the loading rates. The removal rate constants for COD as fitted by the first-order k–C^* model were estimated to be 0.283 and 0.271 m d^?1 for Canna and Heliconia beds, respectively (C^* = 28.1 and 26.7 mg l^?1). Removals of nitrogen (N) and phosphorus (P) were low compared to the loading rates, but removal of total-N was higher in the beds planted with Canna than in beds with Heliconia because of the higher growth rate of Canna. It is concluded that ornamental species like Canna and Heliconia can be used to enhance the aesthetic appearance and hence the public acceptance of wastewater treatment systems in tropical climates. Canna is the preferred species from a treatment perspective because of its more vigorous growth, but since Heliconia has an economic potential as cut flowers may be preferred in many cases.     

Anoxic treatment wetlands for denitrification

Anoxic subsurface flow (SSF) constructed wetlands were evaluated for denitrification using nitrified wastewater. The treatment wetlands utilized a readily available organic woodchip-media packing to create the anoxic conditions. After 2 years in operation, nitrate removal was found to be best described by first-order kinetics. Removal rate constants at 20 °C (k₂₀) were determined to be 1.41–1.30 d^?1, with temperature coefficients (θ) of 1.10 and 1.17, for planted and unplanted experimental woodchip-media SSF wetlands, respectively. First-order removal rate constants decreased as length of operation increased; however, a longer-term study is needed to establish the steady-state values. The hydraulic conductivity in the planted woodchip-media SSF wetlands, 0.13–0.15 m/s, was similar to that measured in an unplanted gravel-media SSF control system.     

Ground level environmental protein concentrations in various ecuadorian environments: Potential uses of aerosolized protein for ecological research

Large quantities of free protein in the environment and other bioaerosols are ubiquitous throughout terrestrial ground level environments and may be integrative indicators of ecosystem status. Samples of ground level bioaerosols were collected from various ecosystems throughout Ecuador, including pristine humid tropical forest (pristine), highly altered secondary humid tropical forest (highly altered), secondary transitional very humid forest (regrowth transitional), and suburban dry montane deforested (suburban deforested). The results explored the sensitivity of localized aerosol protein concentrations to spatial and temporal variations within ecosystems, and their value for assessing environmental change. Ecosystem specific variations in environmental protein concentrations were observed: pristine 0.32 ± 0.09 μg/m³, highly altered 0.07 ± 0.05 μg/m³, regrowth transitional 0.17 ± 0.06 μg/m³, and suburban deforested 0.09 ± 0.04 μg/m³. Additionally, comparisons of intra-environmental differences in seasonal/daily weather (dry season 0.08 ± 0.03 μg/m³ and wet season 0.10 ± 0.04 μg/m³), environmental fragmentation (buffered 0.19 ± 0.06 μg/m³ and edge 0.15 ± 0.06 μg/m³), and sampling height (ground level 0.32 ± 0.09 μg/m³ and 10 m 0.24 ± 0.04 μg/m³) demonstrated the sensitivity of protein concentrations to environmental conditions. Local protein concentrations in altered environments correlated well with satellite-based spectral indices describing vegetation productivity: normalized difference vegetation index (NDVI) (r² = 0.801), net primary production (NPP) (r² = 0.827), leaf area index (LAI) (r² = 0.410). Moreover, protein concentrations distinguished the pristine site, which was not differentiated in spectral indices, potentially due to spectral saturation typical of highly vegetated environments. Bioaerosol concentrations represent an inexpensive method to increase understanding of environmental changes, especially in densely vegetated ecosystems with high canopies or in areas needing high spatial and temporal resolution. Further research to expand understanding of the applicability of bioaerosol concentrations for environmental monitoring is supported by this pilot study.     

Treatment of high-strength wastewater in tropical constructed wetlands planted with Sesbania sesban: Horizontal subsurface flow versus vertical downflow

Treatment of various types of wastewaters is an urgent problem in densely populated areas of many tropical countries. We studied the potential of using Sesbania sesban, an N₂-fixing shrub, in constructed wetland systems for the treatment of high-strength wastewater. A replicated horizontal subsurface flow system and a saturated vertical downflow system was established with planted and unplanted beds to assess the effects of system design and presence of plants on treatment performance. The systems were loaded with a mixture of domestic and pig farm wastewater at three hydraulic loading rates of 80, 160 and 320 mm d^?1. The S. sesban plants grew very well in the constructed wetland systems and produced 17.2–20.2 kg dry matter m^?2 year^?1 with a high nitrogen content. Mass removal rates and removal rate constants increased with loading rate, but at 320 mm d^?1 the effluent quality was unacceptable and hydraulic problems appeared. Mass removal rates and removal rate constants were much higher than reported in other studies probably because of the high-strength wastewater, the high loading rates and the tropical conditions. Planted systems removed pollutants much more efficiently than the unplanted controls. Direct plant uptake constituted only up to 8% of the total-N removal and 2% of the P removal at the lowest loading rate, and was quantitatively of low importance compared to other removal processes. The significant effects of plants were therefore related more to their indirect effects on the removal processes. This study for the first time documents that S. sesban can be used in constructed wetland systems for the treatment of polluted water while at the same time producing a valuable N rich biomass that can be used for animal fodder or soil amendment.     

Does local immersion in thermo-neutral bath influence surface EMG measurements? Results of an experimental trial

This study investigated the effect of water immersion on surface electromyography (EMG) signals recorded from the brachioradial muscle of 11 healthy subjects, both in a dry environment and a thermo-neutral forearm bath (36 °C). EMG measurements were registered in a sitting position, using waterproof electrodes under 3 conditions: relaxed muscle, maximum voluntary isometric contraction (MVC, 1 s, grip test) and 70% of the MVC (5 s). In relaxed muscle, mean EMG values were significantly higher under immersion compared to the dry conditions (dry: 5.4 ± 3.6 μV; water: 19.5 ± 14.9 μV; p = 0.014). In maximum voluntary isometric contraction, there was a significant difference, though not in the same direction (dry: 145.9 ± 58.9 μV; water: 73.2 ± 35.0 μV; p = 0.003). Under 70% MVC, there was no difference between wet and dry conditions (dry: 102.4 ± 75.0 μV; water: 100.4 ± 65.3 μV; p = 0.951). Results suggest that dry and underwater conditions influence EMG readings; however, the results are inconsistent. These findings indicate additional influences on resting muscle activity, as well as MVC. Further measurements with other muscle groups and different types of immersion are needed to clarify conflicting observations.     

Low frequency-low voltage alternating electric current-induced anoxic granulation in biofilm-electrode reactor: A study of granule properties

We characterized anoxic nitrate granules produced by an alternating current biofilm electrode reactor operating at low voltage-low frequency under optimum conditions. Hydrodynamic results revealed that the settling velocity for the granules ranged from 0.12 to 2.85 cm/s, while that of settling velocities ranged from 0.07 to 3.42 cm/s. Granule diameter varied, with a mean mass of 3.65 ± 1.29 mm and corresponding dry mass ranging from 0.52 to 5.64 mg. Roundness ratio of the sampled granules was determined to be 0.78 ± 0.11. Integrity coefficient obtained from a shear strength test was 87.05 ± 2.07% after 2 min and 74.1 ± 4.14% after 5 min. An adhesion test revealed hydrophilic properties of bacteria. The Most probable number (MPN) value was 2.0 × 10⁶ for HDB and 2.0 × 10³ for ADB. An apoptosis assay by flow cytometry confirmed that the majority of cells (87.7%) were viable and non-apoptotic (Annexin V-PI) and dehydrogenase activity was 15.05 ± 1.76 μg TF/mg biomass cm⁻² d. Comparison of seed and granules by ¹H NMR spectra showed different signals in the range of 0.279–1, 1–1.5, and 1.5–7.5 ppm. Therefore, the biofilm in ACBER can be easily granulated and used to generate dense and fast-settling sludge granules.     

Operation of a fixed-bed bioreactor in batch and fed-batch modes for production of inulinase by solid-state fermentation

This work is focused on the inulinase production by solid-state fermentation (SSF) in a fixed-bed reactor (34 cm diameter and 50 cm height) with working capacity of 2-kg of dry substrate operated in batch and fed-batch modes. It was investigated different strategies for feeding the inlet air in the bioreactor (saturated and unsaturated air) as alternative to remove the metabolic heat generated during the microbial growth by evaporative cooling. The kinetic evaluation of the process carried out in batch mode using unsaturated air showed that the evaporative cooling decreasing the mean temperature of the solid-bed, although the enzyme production was lower than that obtained using saturated air. Results showed that maximum enzyme activity (586 ± 63 U gds⁻¹) was obtained in the fed-batch mode using saturated air after 24 h of fermentation. The enzymatic extract obtained by fed-batch mode was characterized and presented optimum temperature and pH in the range of 52–57 °C and 4.8–5.2, respectively. For a temperature range from 40 to 70 °C the enzyme presented decimal reduction time, D-value, ranging from 5748 to 47 h, respectively. For a pH range from 3.5 to 5.5 the enzyme showed good stability, presenting D-values higher than 2622 h. In terms of Michaelis–Mentem parameters were demonstrated that the crude inulinase activity presented higher affinity for substrate sucrose compared to inulin.     

Application of an integrated statistical design to optimize the cold enzyme hydrolysis conditions for ethanol production

Cold enzyme hydrolysis was investigated on the ethanol production by Saccharomyces cerevisiae during simultaneous saccharification and fermentation (SSF) processing. An integrated statistical design, which incorporated single factor design, response surface methodology (RSM) and weighting coefficient method, was used to determine the optimum hydrolysis conditions leading to maximum biomass, ethanol concentration and starch utilization ratio. After the studied ranges of α-amylase, glucoamylase and liquefaction time were identified by single factor design, RSM was used to further optimize the hydrolysis conditions for each objective. The results showed that, under hydrolysis condition optimized with RSM, biomass, ethanol concentration and starch utilization ratio reached 4.401 ± 0.042 × 10⁸ cells/ml, 14.81 ± 0.23% (wt.%) and 94.52 ± 0.53%, respectively. Finally, multi-objective optimization (MOO) was applied to obtain a compromised result of three desirable responses by weighting coefficient methodology. Biomass of 4.331 ± 0.038 × 10⁸ cells/ml, ethanol concentration of 14.12 ± 0.21% (wt.%) and starch utilization ratio of 92.88 ± 0.21% were simultaneous obtained when hydrolysis at pH 5.9 for 114 min with 233 IU/g_starch α-amylase and 778 IU/g_starch glucoamylase. The optimized conditions were shown to be feasible and reliable through verification tests.     

Implementation of a dose–response curve for γ-radiation in the Portuguese population by use of the chromosomal aberration assay

An in vitro dose–response curve following exposure to γ-radiation was determined at the IST/ITN, by use of the chromosomal aberration assay. This is the first study of this kind carried out among the Portuguese population. Un-irradiated and γ-irradiated peripheral blood lymphocytes from 16 healthy donors were cultured. A total of 22,395 metaphases were analyzed for frequency and distribution of dicentrics and centric rings, as a function of the radiation dose. The dose–response data for dicentrics and dicentrics plus centric rings were fitted by use of a linear–quadratic model: Y_dic = (0.0011 ± 0.0006) + (0.0105 ± 0.0035)D + (0.0480 ± 0.0019)D² and Y_{dic + rings} = (0.0011 ± 0.0006) + (0.0095 ± 0.0036)D + (0.0536 ± 0.0020)D². Also, calibration curves related to age and gender were determined, but no significant differences were found. Following the establishment of the dose–response curves, a validation experiment was carried out with three individuals. Real and estimated doses, obtained with the dose–response curves, were in agreement. These results give us confidence to apply both dose–response calibration curves in future biological dosimetry requirements.     

A comparative study of surface and subsurface flow constructed wetlands for treatment of combined sewer overflows: A greenhouse experiment

The use of surface flow (SFCWs) and subsurface flow constructed wetlands (SFCWs) for the treatment of combined sewer overflows was assessed at pilot scale. Synthetic wastewater was applied in three batches with decreasing concentrations to mimic concentration profiles that are obtained in the field during overflow events. Three simulated combined sewer overflows were applied on each wetland. Composite water samples (60 in total) were taken for a period of 8 days to study the removal of total nitrogen (Ntot), NH₄–N, NO₃–N, total COD (CODtot) and total phosphorus. Redox potential, which was monitored at various locations along the wetlands, was more negative in the SSFCWs. In general, removal occurred faster in the SSFCWs and the final concentrations were lower. The removal of Ntot was only 36.6 ± 3.3% in the SFCWs due to nitrification-limiting conditions. The conditions in the SSFCWs, in contrast, seemed to promote Ntot removal (removal efficiency 96.7 ± 1.9%). The removal of P was hampered in both wetland types by reducing conditions. P that was initially removed was released again from the substrates later on. First-order removal rate constants were derived for the removal of both CODtot (SSFCWs: 1.1 ± 0.3 m d^?1; SFCWs: 0.17 ± 0.06 m d^?1) and Ntot (SSFCWs: 0.4 ± 0.1 m d^?1; SFCWs: 1.7 ± 0.5 m d^?1).     

The seed bank in a subtropical freshwater marsh: implications for wetland restoration

Seed bank samples were collected from Huli Marsh, a subtropical shallow water mountainous marsh in Hunan Province, South China. Core samples were divided into upper and lower layers (each 5 cm in depth) and allowed to germinate in three water levels (0, 5 and 10 cm) over a 4-month period. A total of 51 species germinated and the mean density was 9211 ± 7188 seedlings m⁻². In the top 5 cm 41 species and 5747 ± 5111 seedlings m⁻² germinated, whereas 40 species and 3464 ± 3363 seedlings m⁻² did so from 5–10 cm. Germinated seedling density was significantly higher in the upper layer, largely due to differences in eight species. With increasing experimental water depth, less seedlings germinated: respectively, 9788 ± 7157 m⁻², 2050 ± 2412 m⁻² and 1978 ± 2616 m⁻², of 44, 21 and 19 species, submerged under 0, 5 or 10 cm. Seven species could emerge only in 0 water level. Vallisneria natans occurred only in 5 cm water, whereas Ottelia alismoides occurred in 10 cm water. In the vegetation survey of the marsh, 25 species were recorded, which was less than half of the species recorded in the seed bank. The top 10 dominants in the standing vegetation, accounting for 89% of vegetation abundance, represented only 10% in the seed bank. Twenty germinated species that also occurred in the standing vegetation accounted for 56% of the total seed bank. Our observed number of species germinating from a Chinese wetland seed bank is within the range observed elsewhere in the northern hemisphere (15–113 species).