Microbial nitrogen removal of ammonia wastewater in poly (butylenes succinate)-based constructed wetland: effect of dissolved oxygen

Constructed wetland (CW) is popular in wastewater treatment for its prominent advantage of low construction and operation cost. However, the nitrogen removal in conventional CW is usually limited by the low dissolved oxygen (DO) and insufficient electron donor. This paper investigated the nitrogen removal performance and mechanisms in the poly (butylenes succinate)-based CW (PBS-CW) while treating ammonia wastewater under different DO levels. The average DO contents in limited-aeration and full-aeration phases were 1.68 mg L⁻¹ and 5.71 mg L⁻¹, respectively. Results indicated that, with the ammonia nitrogen loading rate of 25 g N m⁻³ day⁻¹, total nitrogen removal ratios in the PBS-CW under the limited-aeration and full-aeration phases were 72% and 99%, respectively. Combined analyses revealed that simultaneous nitrification and denitrification (SND) via nitrite/nitrate were the main microbial nitrogen removal pathways in the aeration phase of the PBS-CW (> 89%). The microbial carrier of biodegradable material was believed to play a significant role in prompting SND performance while dealing with low C/N wastewater. Due to the coexistence of micro-anaerobic zone and carbon supply inside the coated biofilm, the high DO level in the PBS-CW increased the abundance of the nitrifying bacteria (amoA and nxrA), denitrifying bacteria (narG, nirK, nirS, and nosZ), and even anammox bacteria (anammox 16s rRNA). These features are beneficial to many microbial processes which require the simultaneous aerobic, anoxic, and anaerobic environment.

     

Stimulating denitrification in a stream mesocosm with elemental sulfur as an electron donor

The heavy use of fertilizers in agricultural lands can result in significant nitrate (NO₃⁻) loadings to the aquatic environment. We hypothesized that biological denitrification in agricultural ditches and streams could be enhanced by adding elemental sulfur (S^o) to the sediment layer, where it could act as a biofilm support and electron donor. Using a bench-scale stream mesocosm with a bed of S^o granules, we explored NO₃⁻ removal fluxes as a function of the effluent NO₃⁻ concentrations. With effluent NO₃⁻ ranging from 0.5 mg N L⁻¹ to 4.1 mg N L⁻¹, NO₃⁻ removal fluxes ranged from 228 mg N m⁻² d⁻¹ to 708 mg N m⁻² d⁻¹. This is as much as 100 times higher than for agricultural drainage streams. Sulfate (SO₄²⁻) production was high due to aerobic sulfur oxidation. Molecular studies demonstrated that the S^o amendment selected for Thiobacillus species, and that no special inoculum was required for establishing a S^o-based autotrophic denitrifying community. Modeling studies suggested that denitrification was diffusion limited, and advective flow through the bed would greatly enhance NO₃⁻ removal fluxes. Our results indicate that amendment with S^o is an effective means to stimulate denitrification in a stream environment. To minimize SO₄²⁻ production, it may be better to place S^o deeper in the sediment layer.     

Rates, controls and potential adverse effects of nitrate removal in a denitrification bed

Denitrification beds are a simple approach for removing nitrate (NO₃⁻) from a range of point sources prior to discharge into receiving waters. These beds are large containers filled with woodchips that act as an energy source for microorganisms to convert NO₃⁻ to nitrogen (N) gases (N₂O, N₂) through denitrification. This study investigated the biological mechanism of NO₃⁻ removal, its controlling factors and its adverse effects in a large denitrification bed (176 m × 5 m × 1.5 m) receiving effluent with a high NO₃⁻ concentration (>100 g N m⁻³) from a hydroponic glasshouse (Karaka, Auckland, New Zealand). Samples of woodchips and water were collected from 12 sites along the bed every two months for one year, along with measurements of gas fluxes from the bed surface. Denitrifying enzyme activity (DEA), factors limiting denitrification (availability of carbon, dissolved organic carbon (DOC), dissolved oxygen (DO), temperature, pH, and concentrations of NO₃⁻, nitrite (NO₂⁻) and sulfide (S²⁻)), greenhouse gas (GHG) production - as nitrous oxide (N₂O), methane (CH₄), carbon dioxide (CO₂) - and carbon (C) loss were determined. NO₃⁻-N concentration declined along the bed with total NO₃⁻-N removal rates of 10.1 kg N d⁻¹ for the whole bed or 7.6 g N m⁻³ d⁻¹. NO₃⁻-N removal rates increased with temperature (Q₁₀ = 2.0). In laboratory incubations, denitrification was always limited by C availability rather than by NO₃⁻. DO levels were above 0.5 mg L⁻¹ at the inlet but did not limit NO₃⁻-N removal. pH increased steadily from about 6 to 7 along the length of the bed. Dissolved inorganic carbon (C-CO₂) increased in average about 27.8 mg L⁻¹, whereas DOC decreased slightly by about 0.2 mg L⁻¹ along the length of the bed. The bed surface emitted on average 78.58 μg m⁻² min⁻¹ N₂O-N (reflecting 1% of the removed NO₃⁻-N), 0.238 μg m⁻² min⁻¹ CH₄ and 12.6 mg m⁻² min⁻¹ CO₂. Dissolved N₂O-N increased along the length of the bed and the bed released on average 362 g dissolved N₂O-N per day coupled with N₂O emission at the surface about 4.3% of the removed NO₃⁻-N as N₂O. Mechanisms to reduce the production of this GHG need to be investigated if denitrification beds are commonly used. Dissolved CH₄ concentrations showed no trends along the length of the bed, ranging from 5.28 μg L⁻¹ to 34.24 μg L⁻¹. Sulfate (SO₄²⁻) concentrations declined along the length of the bed on three of six samplings; however, declines in SO₄²⁻ did not appear to be due to SO₄²⁻ reduction because S²⁻ concentrations were generally undetectable. Ammonium (NH₄⁺) (range: <0.0007 mg L⁻¹ to 2.12 mg L⁻¹) and NO₂⁻ concentrations (range: 0.0018 mg L⁻¹ to 0.95 mg L⁻¹) were always very low suggesting that anammox was an unlikely mechanism for NO₃⁻ removal in the bed. C longevity was calculated from surface emission rates of CO₂ and release of dissolved carbon (DC) and suggested that there would be ample C available to support denitrification for up to 39 years.This study showed that denitrification beds can be an efficient tool for reducing high NO₃⁻ concentrations in effluents but did produce some GHGs. Over the course of a year NO₃⁻ removal rates were always limited by C and temperature and not by NO₃⁻ or DO concentration.     

Ecology of aquatic Ranunculus communities under the Mediterranean climate

The Iberian Peninsula encompasses more than 80% of the species richness of European aquatic ranunculi. The floristic diversity of the phytocoenosis characterised by aquatic Ranunculus and the main physical–chemical factors of the water were studied in 43 localities of the central Iberian Peninsula. Four aquatic Ranunculus communities are found in most of the aquatic environments. These are species-poor and have an uneven distribution: three species of Batrachium are heterophyllous and their communities are distributed in different aquatic ecosystems on silicated substrates; one species is homophyllous and its community occurs in various aquatic ecosystems with carbonated waters. In the Mediterranean climate, Ranunculus species are present in different habitats, as shown by the results of all the statistical analyses. Ranunculus trichophyllus communities occur in base-rich waters with a high buffering capacity (2273.44 ± 794.57 mg CaCO₃ L⁻¹) and a high concentration of cations (Ca²⁺, 121 ± 33.12 mg L⁻¹; Mg²⁺, 71.64 ± 82.77 mg L⁻¹), nitrates (2.89 ± 4.80 mg L⁻¹), ammonium (2.19 ± 1.36 mg L⁻¹) and sulphates (216.25 ± 218.54 mg L⁻¹). Ranunculus penicillatus communities are found in flowing waters with a high concentration of phosphates (0.48 ± 0.6 mg L⁻¹) and intermediate buffering capacity (683.66 ± 446.76 mg CaCO₃ L⁻¹). Both Ranunculus pseudofluitans and Ranunculus peltatus communities grow in waters with low buffering capacity (R. pseudofluitans, 385.91 ± 209.2 mg CaCO₃ L⁻¹; R. peltatus, 263.3 ± 180.36 mg CaCO₃ L⁻¹), and a low concentration of cations (R. pseudofluitans: Ca²⁺, 12.57 ± 9.42 mg L⁻¹; Mg²⁺, 3.42 ± 1.67 mg L⁻¹; R. peltatus: Ca²⁺, 15 ± 18.26 mg L⁻¹; Mg²⁺, 6.26 ± 8.89 mg L⁻¹) and nutrients (R. pseudofluitans: nitrates, 0.23 ± 0.2 mg L⁻¹; phosphates, 0.09 ± 0.1 mg L⁻¹; R. peltatus: nitrates, 0.19 ± 0.21 mg L⁻¹; phosphates, 0.09 ± 0.12 mg L⁻¹); the first in flowing waters, the latter in still waters.     

Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor

Up-flow oxygen-controlled biofilm reactors equipped with a non-woven fabric support were used as a single reactor system for autotrophic nitrogen removal based on a combined partial nitrification and anaerobic ammonium oxidation (anammox) reaction. The up-flow biofilm reactors were initiated as either a partial nitrifying reactor or an anammox reactor, respectively, and simultaneous partial nitrification and anammox was established by careful control of the aeration rate. The combined partial nitrification and anammox reaction was successfully developed in both biofilm reactors without additional biomass inoculation. The reactor initiated as the anammox reactor gave a slightly higher and more stable mean nitrogen removal rate of 0.35 (± 0.19) kg-N m⁻³ d⁻¹ than the reactor initiated as the partial nitrifying reactor (0.23 (± 0.16) kg-N m⁻³ d⁻¹). FISH analysis revealed that the biofilm in the reactor started as the anammox reactor were composed of anammox bacteria located in inner anoxic layers that were surrounded by surface aerobic AOB layers, whereas AOB and anammox bacteria were mixed without a distinguishable niche in the biofilm in the reactor started as the partial nitrifying reactor. However, it was difficult to efficiently maintain the stable partial nitrification owing to inefficient aeration in the reactor, which is a key to development of the combined partial nitrification and anammox reaction in a single biofilm reactor.     

Autotrophic denitrification using hydrogen generated from metallic iron corrosion

Hydrogenotrophic denitrification was demonstrated using hydrogen generated from anoxic corrosion of metallic iron. For this purpose, a mixture of hydrogenated water and nitrate solution was used as reactor feed. A semi-batch reactor with nitrate loading of 2000 mg m⁻³ d⁻¹ and hydraulic retention time (HRT) of 50 days produced effluent with nitrate concentration of 0.27 mg N L⁻¹ (99% nitrate removal). A continuous flow reactor with nitrate loading of 28.9 mg m⁻³ d⁻¹ and HRT of 15.6 days produced effluent with nitrate concentration of ∼0.025 mg N L⁻¹ (95% nitrate removal). In both cases, the concentration of nitrate degradation by-products, viz., ammonia and nitrite, were below detection limits. The rate of denitrification in the reactors was controlled by hydrogen availability, and hence to operate such reactors at higher nitrate loading rates and/or lower HRT than reported in the present study, hydrogen concentration in the hydrogenated water must be significantly increased.     

Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure

In this study, the performance of partial nitrification via nitrite and microbial community structure were investigated and compared in two sequencing batch reactors (SBR) with different dissolved oxygen (DO) levels. Both reactors achieved stable partial nitrification with nitrite accumulation ratio of above 95% by using real-time aeration duration control. Compared with high DO (above 3 mg/l on average) SBR, simultaneous nitrification and denitrification (SND) via nitrite was carried out in low DO (0.4–0.8 mg/l) SBR. The average efficiencies of SND in high DO and low DO reactor were 7.7% and 44.9%, and the specific SND rates were 0.20 and 0.83 mg N/(mg MLSS h), respectively. Low DO did not produce sludge with poorer settling properties but attained lower turbidities of the effluent than high DO. Fluorescence in situ hybridization (FISH) analysis in both the reactors showed that ammonia-oxidizing bacteria (AOB) were the dominant nitrifying bacteria and nitrite-oxidizing bacteria (NOB) did not be recovered in spite of exposing nitrifying sludge to high DO. The morphology of the sludge from both two reactors according to scanning electron microscope indicated that small rod-shaped and spherical clusters were dominant, although filamentous bacteria and few long rod-shaped coexisted in the low DO reactor. By selecting properly DO level and adopting process control method is not only of benefit to the achievement of novel biological nitrogen removal technology, but also favorable to sludge population optimization.     

Performance of a pilot-scale sewage treatment: an up-flow anaerobic sludge blanket (UASB) and a down-flow hanging sponge (DHS) reactors combined system by sulfur-redox reaction process under low-temperature conditions

Performance of a wastewater treatment system utilizing a sulfur-redox reaction of microbes was investigated using a pilot-scale reactor that was fed with actual sewage. The system consisted of an up-flow anaerobic sludge blanket (UASB) reactor and a down-flow hanging sponge (DHS) reactor with a recirculation line. Consequently, the total COD_Cr (465 ± 147 mg L⁻¹; total BOD of 207 ± 68 mg L⁻¹) at the influent was reduced (70 ± 14 mg L⁻¹; total BOD of 9 ± 2 mg L⁻¹) at the DHS effluent under the conditions of an overall hydraulic retention time of 12 h, a recirculation ratio of 2, and a low-sewage temperature of 7.0 ± 2.8 °C. A microbial analysis revealed that sulfate-reducing bacteria contributed to the degradation of organic matter in the UASB reactor even in low temperatures. The utilized sulfur-redox reaction is applicable for low-strength wastewater treatment under low-temperature conditions.     

The treatment of cheese whey wastewater by sequential anaerobic and aerobic steps in a single digester at pilot scale

The treatment of reconstituted whey wastewater was performed in a 400 L digester at 20 °C, with an anaerobic digestion step, followed by a step of aerobic treatment at low oxygen concentration in the same digester. In a first set of 48 cycles, total cycle time (T_C) of 2, 3 and 4 days were tested at varying organic loading rates (OLR). The COD removal reached 89 ± 4, 97 ± 3 and 98 ± 2% at T_C of 2, 3 and 4 days and OLR of 0.56, 1.04 and 0.78 gCOD L⁻¹ d⁻¹, respectively. The activity of the biomass decreased for the methanogenic population, while increasing by 400% for the acidogens, demonstrating a displacement in the predominant trophic group in the biomass bed. A second set of 16 cycles was performed with higher soluble oxygen concentration in the bulk liquid (0.5 mg L⁻¹) during the aerobic treatment at a T_C of 2 days and an OLR of 1.55 gCOD L⁻¹ d⁻¹, with a soluble COD removal of 88 ± 3%. The biomass specific activities showed a compartmentalization of the trophic group with methanogenic activity maintained in the biomass bed and a high acidogenic activity in the suspended flocs.     

Effects of hydraulic loading rate on pollutants removal by a deep subsurface wastewater infiltration system

The subsurface wastewater infiltration (SWI) system proved to be an effective and low-cost technique for decentralized sewage treatment in areas without adequate domestic treatment facilities. Field-scale experiments were conducted through a deep SWI system, with effective depth of 1.5 m, under hydraulic loading rates of 0.040, 0.065, 0.081 and 0.10 m³/m² d. Taking the hydraulic and treatment efficiencies into consideration, the hydraulic loading rate of 0.081 m³/m² d was recommended. Under this condition, NH₃-N, TN, and COD removal efficiencies were 86.2 ± 3.0, 80.7 ± 1.9 and 84.8 ± 2.1%, respectively. In the effluent, NH₃-N concentration declined to 2.3-4.4 mg/L, accounting for 63.2-65.6% of TN. NO₃-N concentration increased from 0.2 to 0.3 mg/L in the influent to 2.0-2.5 mg/L in the effluent. The nitrifying bacteria number declined with increased depth, while the amount of denitrifying bacteria increased. The analysis of results about the nitrifying and denitrifying bacteria distribution indicated that the most effective ranges for nitrification and denitrification process were 0.3-0.7 m and 0.7-1.5 m, respectively.     

Biomass production and nitrogen and phosphorus removal by the green alga Neochloris oleoabundans in simulated wastewater and secondary municipal wastewater effluent

Biomass productivity of 350 mg DCW L⁻¹ day⁻¹ with a final biomass concentration of 3.15 g DCW L⁻¹ was obtained with Neochloris oleoabundans grown in artificial wastewater at sodium nitrate and phosphate concentrations of 140 and 47 mg L⁻¹, respectively, with undetectable levels of residual N and P in effluents. In secondary municipal wastewater effluents enriched with 70 mg N L⁻¹, the alga achieved a final biomass concentration of 2.1 g DCW L⁻¹ and a biomass productivity of 233.3 mg DCW L⁻¹ day⁻¹. While N removal was very sensitive to N:P ratio, P removal was independent of N:P ratio in the tested range. These results indicate that N. oleoabundans could potentially be employed for combined biofuel production and wastewater treatment.     

Evaluation of a lab-scale tidal flow constructed wetland performance: Oxygen transfer capacity, organic matter and ammonium removal

Oxygen transfer capacity and removal of ammonium and organic matter were investigated in this study to evaluate the performance of a lab-scale tidal flow constructed wetland. Average oxygen supply under tidal operation (350 g m⁻² d⁻¹) was much higher than in conventional constructed wetlands (<100 g m⁻² d⁻¹), resulting in enhanced removal of BOD₅ and NH₄⁺. Theoretical oxygen demand from BOD₅ removal and nitrification was approximately matched by the measured oxygen supply, which indicated aerobic consumption of BOD₅ and NH₄⁺ under tidal operation. When BOD₅ removal increased from 148 g m⁻² d⁻¹ to 294 g m⁻² d⁻¹, neither exhausted oxygen from the aggregate matrix during feeding period (111 g m⁻² d⁻¹) nor effluent dissolved oxygen (DO) concentration (2.8 mg/L) changed significantly, demonstrating that the oxygen transfer potential of the treatment system had not been exceeded. However, even though DO had not been exhausted, inhibition of nitrification was observed under high BOD loading. The loss of nitrification was attributed to excessive heterotrophic biofilm growth believed to induce oxygen transfer limitations or oxygen competition in thickened biofilms.     

Electricity generation in single-chamber microbial fuel cells using a carbon source sampled from anaerobic reactors utilizing grass silage

Production of electricity from samples obtained during anaerobic digestion of grass silage was examined using single-chamber air-cathode mediator-less microbial fuel cells (MFCs). The samples were obtained from anaerobic reactors at start-up conditions after 3 and 10 days of operation under psychrophilic (15 °C) and mesophilic (37 °C) temperatures. Electricity was directly produced from all samples at a concentration of 1500 mg COD L⁻¹. Power density obtained from the samples, as a sole carbon source, ranged from 56 ± 3 W m⁻³ to 31 ± 1 W m⁻³ for the mesophilic and psychrophilic samples, respectively. Coulombic efficiencies ranged from 18 ± 1% to 12 ± 1% for the same samples. The relationship between the maximum voltage output and initial COD concentration appeared to follow saturation kinetics at the external resistance of 217 Ω. Chemical oxygen demand (COD) removal was over 90% and total phenolics removal was in the range of 30-75% for all samples tested, with a standard amount of 60 mg L⁻¹ total phenolics removed for every sample. Our results indicate that generating electricity from solution samples of anaerobic reactors utilizing grass silage is possible, opening the possibility for combination of anaerobic digestion with MFC technology for energy generation.     

A control strategy of partial nitritation in a fixed bed biofilm reactor

To achieve an appropriate mixture of ammonium and nitrite for anaerobic ammonium oxidation (ANAMMOX), 50% partial nitritation was optimized in a fixed bed biofilm reactor treating synthetic wastewater. Results suggested that 50% partial nitritation could be achieved by stepwise increases of influent NH₄⁺-N at pH of 7.8 ± 0.2, temperature of 30 ± 1 °C and dissolved oxygen (DO) of 0.5-0.8 mg l⁻¹. Hydraulic retention time (HRT) and influent alkalinity did significantly affect partial nitritation. At HRT 12 h, 50% partial nitritation could be kept stable, regardless of influent NH₄⁺-N variation, by controlling the influent HCO₃⁻/NH₄⁺ molar ratio at 1:1. The fluorescent in situ hybridization (FISH) results indicated the abundance of evolution of ammonia-oxidizing bacteria (AOB) and the nitrite-oxidizing bacteria (NOB) coincided well with the performance of partial nitritation. Furthermore, the AOB were highly affiliated with Nitrosomonas spp. and Nitrosospira spp. dominated (64.1%) in the biofilm with a compact structure during the stable 50% partial nitritation period.     

Bio-kinetic analysis on treatment of textile dye wastewater using anaerobic batch reactor

An anaerobic digestion technique was applied to textile dye wastewater aiming at the colour and COD removal. Pet bottles of 5 L capacity were used as reactor which contains methanogenic sludge of half a liter capacity which was used for the treatment of combined synthetic textile dye and starch wastewater at different mixing ratios of 20:80, 30:70, 40:60, 50:50 and 60:40 with initial COD concentrations as 3520, 3440, 3360, 3264 and 3144 mg L⁻¹, respectively. The reactor was maintained at room temperature (30 ± 3 °C) with initial pH of 7. The maximum COD and colour removal were 81.0% and 87.3% at an optimum mixing ratio of 30:70 of textile dye and starch wastewaters. Both Monod’s and Haldane’s models were adopted in this study. The kinetic constants of cell growth under Haldane’s model were satisfactory when compared to Monod’s model. The kinetic constants obtained by Haldane’s model were found to be in the range of μ_max = 0.037-0.146 h⁻¹, K_s = 651.04-1372.88 mg L⁻¹ and K_i = 5681.81-18727.59 mg L⁻¹.     

Influence of food colorant and initial COD concentration on the efficiencies of micro-aerobic sequencing batch reactor (micro-aerobic SBR) for casein recovery under non-sterile condition by Lactobacillus casei TISTR 1500

The acid biocoagulants produced from non-sterile lactic acid fermentation by Lactobacillus casei TISTR 1500 were used to settle colloidal protein, mainly casein, at the isoelectric point in dairy effluent prior to secondary treatment. High concentration of azo dye (Ponceau 4R) in the dairy wastewater and the stress of starvation decreased the efficiencies of the micro-aerobic SBR. Consequently, low casein recovery obtained and organic removal suffered a decline. The number of lactic acid bacteria (LAB) also declined from log 7.4 to log 5.30 in the system fed with 400 mg L⁻¹ of the dye containing wastewater. The recovery of the system, however, showed that 25,000 mg COD L⁻¹ influent with 200 mg L⁻¹ of the dye maintained the growth of LAB in the range of log 7.74–8.12, with lactic and acetic production (2597 and 197 mg L⁻¹) and 83% protein removal. The results in this study suggested that the inhibitory effects were compensated with high organic content feeding.     

Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1

Effects of heavy metals on aerobic denitrification have been poorly understood compared with their impacts on anaerobic denitrification. This paper presented effects of four heavy metals (Cd(II), Cu(II), Ni(II), and Zn(II)) on aerobic denitrification by a novel aerobic denitrifying strain Pseudomonas stutzeri PCN-1. Results indicated that aerobic denitrifying activity decreased with increasing heavy metal concentrations due to their corresponding inhibition on the denitrifying gene expression characterized by a time lapse between the expression of the nosZ gene and that of the cnorB gene by PCN-1, which led to lower nitrate removal rate (1.67∼6.67 mg L⁻¹ h⁻¹), higher nitrite accumulation (47.3∼99.8 mg L⁻¹), and higher N₂O emission ratios (5∼283 mg L⁻¹/mg L⁻¹). Specially, promotion of the nosZ gene expression by increasing Cu(II) concentrations (0∼0.05 mg L⁻¹) was found, and the absence of Cu resulted in massive N₂O emission due to poor synthesis of N₂O reductase. The inhibition effect for both aerobic denitrifying activity and denitrifying gene expression was as follows from strongest to least: Cd(II) (0.5∼2.5 mg L⁻¹) > Cu(II) (0.5∼5 mg L⁻¹) > Ni(II) (2∼10 mg L⁻¹) > Zn(II) (25∼50 mg L⁻¹). Furthermore, sensitivity of denitrifying gene to heavy metals was similar in order of nosZ > nirS ≈ cnorB > napA. This study is of significance in understanding the potential application of aerobic denitrifying bacteria in practical wastewater treatment.

     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

A simple method for quantifying biomass cell and polymer distribution in biofilms

Biofilms are ubiquitous and play an essential role in both environmental processes and hospital infections. Standard methods are not capable of quantifying biomass concentration in dilute suspensions. Furthermore, standard techniques cannot differentiate biomass composition. In this study, a user-friendly technique was developed for measuring biomass cell and polymer content in detached biofilms using a standard coulter counter. The method was demonstrated for an environmentally relevant strain of Pseudomonas aeruginosa (Schroeter) Migula grown in a bioreactor and also for a medically relevant strain of P. aeruginosa (PAO1) grown on standard growth pegs. Results were compared and validated by standard assays, including EPA method 1684 for measuring biomass, microscopic direct counts, and a crystal violet staining assay. The minimum detection limit for the coulter counter method (0.07 mg-biomass L^− 1) was significantly lower than the EPA method 1684 (1.9 ± 0.4 mg/L) and the crystal violet assay (1.1 ± 0.2 mg L^− 1). However, the coulter counter method is limited to dilute biomass samples (below 204 ± 16 mg L^− 1) due to clogging of the aperture tube. While biomass measurements are useful, the major advantage of the coulter counter method is the ability to directly determine EPS, cell, and aggregate fractions after mild chemical treatment. The rapid technique (4–5 min per sample) was used to measure biomass fractions in dispersed P. aeruginosa (Schroeter) and PAO1 biofilms. This technique will be critical for understanding biofilm formation/dispersal.     

Phagocytic activity,respiratory burst,cytoplasmic free-Ca concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress

The aim of this study was to investigate the cellular toxicity of copper-induced injury to the black tiger shrimp Penaeus monodon. The 24 h, 48 h, 72 h and 96 h LC₅₀ (median lethal concentration) of Cu²⁺ on P. monodon (11.63 ± 1.14 g) were found to be 3.49, 1.54, 0.73 and 0.40 mg L^− 1, respectively. Total haemocyte count (THC), phagocytic activity, respiratory burst (RB), cytoplasmic free-Ca²⁺ (cf-Ca²⁺) concentration and apoptotic cell ratio of shrimp were determined after exposure to different concentrations of Cu²⁺ (0, 0.05, 0.5, 1.5 and 3.5 mg L^− 1) for 0, 6, 12, 24 and 48 h. There was no significant effect on the analytic indicator of shrimp exposed to 0.05 mg L^− 1 Cu²⁺. THC decreased after Cu-exposure to 0.5 mg L^− 1 for 48 h, 1.5 mg L^− 1 for 24 h and 3.5 mg L^− 1 for 12 h. Phagocytic activity decreased in P. monodon following 48 h exposure to 3.5 mg L^− 1 Cu²⁺. RB was induced after 6 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. cf-Ca²⁺ concentration increased after 48 h exposure to 0.5 mg L^− 1 Cu²⁺, and 12 h exposure to 1.5 and 3.5 mg L^− 1 Cu²⁺. The percentage of apoptotic cells increased to 9.5%, 16.3% and 18.6% respectively following 48 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. These results indicate that Cu can induce oxidative stress, elevation of cf-Ca²⁺ and cell apoptosis, and inhibit phagocytic activity in the shrimp P. monodon, and the lethal injury of Cu²⁺ to P. monodon may be mainly due to the sharp reduction of THC caused by ROS-induced apoptosis.