Ex situ bioremediation of pyrene contaminated soil in bio-slurry phase reactor operated in periodic discontinuous batch mode: Influence of bioaugmentation

Ex situ treatment of simulated pyrene-contaminated soil was studied in bio-slurry phase reactors operated in periodic discontinuous batch mode under anoxic–aerobic–anoxic–anoxic microenvironment. Experiments were performed in six different bio-slurry phase reactors (retention time of 120 h; soil loading rate of 20 kg soil/m³-day; operating temperature at 28±2 °C) by varying substrate concentration (substrate loading rate (SLR), 0.12, 0.24 and 0.36 g pyrene/kg soil-day) and bioaugmentation application (domestic sewage inoculum; CFU—2×10⁶). The performance of slurry phase reactors was found to be dependent on the applied SLR and application of bioaugmentation (domestic sewage as augmented inoculum). Control reactor (killed control) showed only 6% of pyrene degradation while the non-augmented reactor showed an efficiency of 34% (substrate degradation rate (SDR)—0.0165 g pyrene/kg soil-day). In the case of augmented reactors, the system operated with low SLR showed a pyrene degradation efficiency of almost 90% (SDR—0.04 g pyrene/kg soil-day) and the reactor with high SLR showed 50% (SDR—0.025 g pyrene/kg soil-day) of pyrene degradation indicating the dependence of performance on the substrate concentration. Colony forming units (CFUs) variation was in good agreement with the performance of the reactors with respect to pyrene degradation. On the whole, pyrene degradation rate was greater in the augmented reactors compared to non-augmented reactors.     

Degradation of di-ethylhexyl phthalate (DEHP) in bioslurry phase reactor and identification of metabolites by HPLC and MS

Di-ethylhexyl phthalate (DEHP) belongs to the class of phthalate esters and is used as an additive in many products including plastics, paints and inks or as a solvent in industrial formulations. However, it is used mostly for its plasticizing ability in polyvinyl chloride (PVC) products, in which it is often added in concentrations exceeding 40% by mass. DEHP is one of the more recalcitrant phthalate esters, which has xeno-estrogenic, carcinogenic and mutagenic effects. Five different bioslurry reactors were operated under different conditions to study the degradation of DEHP (1 mg g⁻¹ soil) in soil. The process performance was assessed by monitoring DEHP concentration periodically using high performance liquid chromatography (HPLC). The ongoing biological process was monitored by analyzing pH, oxidation–reduction potential (ORP), dissolved oxygen (DO), oxygen uptake rate (OUR) and colony forming units (CFU) for every 24 h. More than 90% degradation was observed within 12 days of the cycle period in the augmented reactors. Metabolites formed during the degradation of DEHP in the slurry phase reactor were identified and the pathway was also established. The degradation process was found to follow zero-order kinetic model.     

Influence of soil-water ratio on the performance of slurry phase bioreactor treating herbicide contaminated soil

The influence of soil-water ratio was studied on the performance of the slurry phase bioreactor operated in sequencing batch mode (anoxic-aerobic-anoxic microenvironments) during the bioremediation of soil contaminated with pendimethalin. The performance of the reactors was evaluated at different soil-water ratios (1:5-1:25; at soil loading rate (60 kg of soil/cum-day to 12 kg of soil/cum-day)) keeping the loading rate of pendimethalin constant (133.2 g/kg of soil-day) in six reactors and variable (66.6 g/kg of soil-day to 166.6 g/kg of soil-day) in other four reactors. At 1:20 soil-water ratio, the slurry phase system showed enhanced degradation of substrate (629 microg pendimethalin/g soil). The removal efficiency of pendimethalin in the reactors was dependent on the mass-transfer rates of the substrate from the soil to the aqueous phase. Soil-water ratio and substrate loading rates showed significant influence on the substrate portioning, substrate degradation efficiency and substrate desorption rate.     

Improving an on-line feeding strategy for fed-batch cultures of hybridoma cells by dialysis and `Nutrient-Split'-feeding

The concept of the feeding strategy was to minimise the formation of inhibiting metabolites and to increase the yield of monoclonal antibodies in fed-batch cultures of hybridoma cells by a balanced supply of substrates. A process control system based on fieldbus technology was used for monitoring and control. External program routines were implemented to control dissolved oxygen (DO) and to calculate the oxygen uptake rate (OUR) and cumulative oxygen consumption (COC) simultaneously. A concentrated feed solution was supplied according to the off-line estimated stoichiometric ratio between oxygen and glucose consumption (GC). Feeding was initiated automatically when the OUR decreased due to substrate limitation. The antibody concentration increased three-fold compared to the conventional batch culture by applying this strategy. But it was not possible to avoid inhibition by ammonia during the fed-batch phase. This was accomplished by the use of a dialysis membrane. Dialysis fed-batch cultures were performed in a membrane dialysis reactor with a `nutrient-split' feeding strategy, where concentrated medium is fed to the cells and toxic metabolites are removed into a buffer solution. This resulted in a ten-fold increase of the antibody concentration compared to the batch. Amino acid concentrations were analysed to identify limiting conditions during the cultivation and to analyse the performance of the nutrient supply in the fed-batch and dialysis fed-batch.     

Simultaneous nitrification and denitrification using stored substrate (PHB) as the electron donor in an SBR

The potential for PHB (poly-beta-hydroxybutyrate) to serve as the electron donor for effective simultaneous nitrification and denitrification (SND) was investigated in a 2-L sequencing batch reactor (SBR) using a mixed culture and acetate as the organic substrate. During the feast period (i.e., acetate present), heterotrophic respiration activity was high and nitrification was prevented due to the inability of nitrifying bacteria to compete with heterotrophs for oxygen. Once acetate was depleted the oxidation rate of PHB was up to 6 times slower than that of soluble acetate and nitrification could proceed due to the decreased competition for oxygen. The slow nature of PHB degradation meant that it was an effective substrate for SND, as it was oxidised at a similar rate to ammonium and was therefore available for SND throughout the entire aerobic period. The percentage of nitrogen removed via SND increased at lower DO concentrations during the famine period, with up to 78% SND achieved at a DO concentration of 0.5 mg L(-1). However, the increased percentage of SND at a low DO concentration was compromised by a 2-times slower rate of nitrogen removal. A moderate DO concentration of 1 mg L(-1) was optimal for both SND efficiency (61%) and rate (4.4 mmol N x Cmol x(-1) x h(-1)). Electron flux analysis showed that the period of highest SND activity occurred during the first hour of the aerobic famine period, when the specific oxygen uptake rate (SOUR) was highest. It is postulated that a high SOUR due to NH(4) (+) and PHB oxidation decreases oxygen penetration into the floc, creating larger zones for anoxic denitrification. The accumulation of nitrate towards the end of the SND period showed that SND was finally limited by the rate of denitrification. As PHB degradation was found to follow first-order kinetics (df(PHB)/dt = -0.19 x f(PHB)), higher PHB concentrations would be expected to drive SND faster by increasing the availability rate of reducing power and reducing penetration of oxygen into the floc, due to the corresponding increased SOUR. Process control techniques to accumulate higher internal PHB concentrations to improve PHB-driven SND are discussed.     

Feasibility of nitrification/denitrification in a sequencing batch biofilm reactor with liquid circulation applied to post-treatment

An investigation was performed on the biological removal of ammonium nitrogen from synthetic wastewater by the simultaneous nitrification/denitrification (SND) process, using a sequencing batch biofilm reactor (SBBR). System behavior was analyzed as to the effects of sludge type used as inoculum (autotrophic/heterotrophic), wastewater feed strategy (batch/fed-batch) and aeration strategy (continuous/intermittent). The presence of an autotrophic aerobic sludge showed to be essential for nitrification startup, despite publications stating the existence of heterotrophic organisms capable of nitrifying organic and inorganic nitrogen compounds at low dissolved oxygen concentrations. As to feed strategy, batch operation (synthetic wastewater containing 100 mg COD/L and 50 mg N-NH(4)(+)/L) followed by fed-batch (synthetic wastewater with 100 mg COD/L) during a whole cycle seemed to be the most adequate, mainly during the denitrification phase. Regarding aeration strategy, an intermittent mode, with dissolved oxygen concentration of 2.0mg/L in the aeration phase, showed the best results. Under these optimal conditions, 97% of influent ammonium nitrogen (80% of total nitrogen) was removed at a rate of 86.5 mg N-NH(4)(+)/Ld. In the treated effluent only 0.2 mg N-NO(2)(-)/L,4.6 mg N-NO(3)(-)/L and 1.0 mg N-NH(4)(+)/L remained, demonstrating the potential viability of this process in post-treatment of wastewaters containing ammonium nitrogen.     

Long-term operation of slurry bioreactor for decomposition of food wastes

A pilot scale slurry bioreactor was used for the treatment of food wastes. Food wastes were continuously added (750 g wet weight per day) into the reactor and successfully decomposed to inorganic carbon without intermittent removal of suspended solids. During operation for 90 days, 91% reduction of food wastes was achieved. Microorganisms actively grew during the initial 20 days of operation, and reached a stationary phase with a cell concentration of around 5 x 10(10) cells ml(-1), which indicated that food waste was utilized as a respiratory substrate during this phase. Using data for time variation of dissolved oxygen, the oxygen requirement for decomposition of food wastes was estimated to be 5.0 g O2 g(-1) dry weight of food wastes.     

Dynamic response of naphthalene biodegradation in a continuous flow soil slurry reactor

Periodic perturbations were used to evaluate the system stability and robustness of naphthalene biodegradation in a continuous flow stirred tank reactor (CSTR) containing a soil slurry. The experimental design involved perturbing the test system using a sinusoidal input either of naphthalene or non-naphthalene organic carbon at different frequencies during steady state operation of the reactors. The response of the test system was determined by using time series off-gas analysis for naphthalene liquid phase concentration and degradation, total viable cell counts, and gene probe analysis of naphthalene degradative genotype, and by batch mineralization assays.Naphthalene biodegradation rates were very high throughout the experimental run (95 to >99% removed) resulting in very low or undetectable levels of naphthalene in the off-gas and reactor effluent. Attempts to reduce the rate of naphthalene biotransformation by either reducing the reactor temperature from 20°C to 10°C or the dissolved oxygen level (>1 mg/L) were unsuccessful. Significant naphthalene biodegradation was observed at 4°C. While variable, the microbial community as measured by population densities was not significantly affected by temperature changes. In terms of naphthalene biotransformation, the system was able to adapt readily to all perturbations in the reactor.Department of Chemical EngineeringDepartment of Microbiology and The Graduate Program in EcologyDepartment of Civil Engineering, New Orleans University     

Biodegradation of phenol and m-cresol in a batch and fed batch operated internal loop airlift bioreactor by indigenous mixed microbial culture predominantly Pseudomonas sp

An internal loop airlift reactor (ILALR) is developed and studied for biodegradation of phenol/m-cresol as single and dual substrate systems under batch and fed batch operation using an indigenous mixed microbial strain, predominantly Pseudomonas sp. The results showed that the culture could degrade phenol/m-cresol completely at a maximum concentration of 600mgl(-1) and 400mgl(-1), respectively. Batch ILALR study has revealed that phenol has been preferentially degraded by the microbial culture rather than m-cresol probably owing to the toxic effect of the later. Sum kinetic model evaluated the interaction between the phenol/m-cresol in dual substrate system, which resulted in a high coefficient of determination (R(2)) value >0.98). The fed batch results showed that the strain was able to degrade phenol/m-cresol with maximum individual concentrations 600mgl(-1) each in 26h and 37h, respectively. Moreover for fed batch operation, degradation rates increased with increase in feed concentration without any lag in the degradation profile.     

Production of gluconic acid from glucose by Aspergillus niger: growth and non-growth conditions

Batch fermentation of glucose to gluconic acid was conducted using Aspergillus niger under growth and non-growth conditions using pure oxygen and air as a source of oxygen for the fermentation in 2 and 5 l stirred tank reactors (batch reactor). Production of gluconic acid under growth conditions was conducted in a 5 l batch reactor. Production and growth rates were higher during the period of supplying pure oxygen than that during supplying air, and the substrate consumption rate was almost constant. For the production of gluconic acid under non-growth conditions, conducted in the 2 l batch reactor, the effect of the pure oxygen flow rate and the biomass concentration on the gluconic acid production was investigated and an empirical equation suggested to show the dependence of the production rate r_p on the biomass concentration C_x and oxygen flow rate Q, at constant operating conditions (30 °C, 300 rpm and pH 5.5). Biomass concentration had a positive effect on the production rate r_p, and the effect of Q on r_p was positive at high biomass concentrations.     

Performance of batch membrane reactor: Glycerol-3-phosphate synthesis coupled with adenosine triphosphate regeneration.

Glycerol-3-phosphate (G3P) was synthesized from glycerol using glycerol kinase (GK). This reaction requires adenosine triphosphate (ATP) and was coupled with the ATP regeneration reaction using acetate kinase (AK) in a batch-operated ultrafiltration hollow-fiber reactor. By taking into consideration the dynamic nature of the bioreactor performance under non-steady-state conditions, a model for the performance of a batch membrane reactor for G3P synthesis coupled with ATP regeneration was developed and studied. The simulation results showed good agreement with the experimental results. The simulation studies have provided some insight into the process dynamics of the coupled reactions in the reactor system studied. For the reactor operational model used, in which the enzymes are retained in the shell side and the substrates are also initially placed in the shell side, it was found that the substrate concentration in the lumen side increased to a level higher than that in the shell side, and a backdiffusion occurred from the lumen side to the shell side during reactor operation. The ratio of the reaction rate to diffusion rate goes through a sharp peak during the time that the direction of diffusion is reversed. For another reactor operational model, in which the substrates were initially placed in the lumen side and enzymes were retained in the shell side, it was found that the rate-controlling step between the reaction and diffusion was switched during the reactor operation. Initially, the reaction rate increased while the diffusion rate was high and the substrate concentrations increased in the shell side. The ratio of reaction rate to diffusion rate increased to a maximum and remained at a constant level as the diffusion rate decreased to a low level due to the nonlinear characteristics of mass transfer process. This study provides information that is useful for optimization of batch membrane enzyme reactor operation and for a fed-batch-type process with an intermittent feeding strategy for efficient use of substrates.     

Growth and substrate consumption of Nitrobacter agilis cells immobilized in carrageenan: part 2. Model evaluation

A dynamic model that predicts substrate and biomass concentration profiles across gel beads and from that the overall substrate consumption rate by the gel beads containing growing cells was evaluated with immobilized Nitrobacter agilis cells in an airlift loop reactor with oxygen as the limiting substrate. The model predictions agreed well with the observed oxygen consumption rates at three different liquid phase oxygen concentrations. Image analysis showed that 90% of the immobilized cells after 42 days of cultivation was situated in the outer shells in a film of 140 mum, while the bead radius was about 1 mm. The maximum biomass concentration in the outmost film of 56 mum was 11 kg . m(-3) gel.     

Influence of the mode of reactor operation on the rate and yield of fermentation of the model solid substrate,wheat bran,by a microbial consortium under non-axenic anaerobic conditions

Summary Anaerobic fermentation of wheat bran, a model solid substrate, was conducted under non-axenic conditions, in two reactors operated under different modes, all other conditions being strictly identical. The first reactor was a completely-mixed batch reactor. The second reactor was a percolator into which the liquid phase was recirculated in closed loop through the solid substrate acting as a stationary bed. The final yield of fermentation was obtained after 27 days in the completely-mixed batch reactor, and after 14 days only in the percolator. Scanning electron microscopy revealed that numerous micro-organisms adhered to the solid substrate acting as a carrier in the percolator, whereas only very few micro-organisms adhered to the solid substrate in the completely-mixed batch reactor. The results show that obtaining a durable direct contact between micro-organisms and their solid substrate improves the rate of solids degradation.     

Removal of ammonium via simultaneous nitrification-denitrification nitrite-shortcut in a single packed-bed batch reactor

A polyurethane packed-bed-biofilm sequential batch reactor was fed with synthetic substrate simulating the composition of UASB reactor effluents. Two distinct ammonia nitrogen concentrations (125 and 250 mg l(-1)) were supplied during two sequential long-term experiments of 160 days each (320 total). Cycles of 24h under intermittent aeration for periods of 1h were applied, and ethanol was added as a carbon source at the beginning of each anoxic period. Nitrite was the main oxidized nitrogen compound which accumulated only during the aerated phases of the batch cycle. A consistent decrease of nitrite concentration started always immediately after the interruption of oxygen supply and addition of the electron donor. Removal to below detection limits of all nitrogen soluble forms was always observed at the end of the 24h cycles for both initial concentrations. Polyurethane packed-bed matrices and ethanol amendments conferred high process stability. Microbial investigation by cloning suggested that nitrification was carried out by Nitrosomonas-like species whereas denitrification was mediated by unclassified species commonly observed in denitrifying environments. The packed-bed batch bioreactor favored the simultaneous colonization of distinct microbial groups within the immobilized microbial biomass. The biofilm was capable of actively oxidizing ammonium and denitrification at high ratios in intermittent intervals within 24h cycles.     

Aerobic degradation of the azo dye acid red 151 in a sequencing batch biofilter

The azo dye acid red 151 (AR151) was aerobically biodegraded in a sequencing batch biofilter packed with a porous volcanic rock. AR151 was used as the sole source of carbon and energy for acclimated microorganisms. Acclimation was followed using the degradation time and the oxygen uptake rate. A maximal oxygen uptake rate of 0.5 mg O(2)/(lmin) was obtained. Mineralization studies showed that 73% (as carbon) of the initial azo dye was transformed to CO(2) by the consortia. A maximal substrate degradation rate of 247 mg AR151/(l(reactor)d) was obtained. Color removal was up to 99% using an initial concentration of 50 mg AR151/l. Anaerobic tests suggested that in the interior of the porous material, anaerobic biotransformations can occur, contributing from 14% to 16% of the decoloration of the azo dye.     

Bioremediation of tetryl-contaminated soil using sequencing batch soil slurry reactor

A laboratory study was conducted to determine whether tetryl (2,4,6-trinitrophenlymethylnitramine) contaminated soil could be bioremediated using a sequencing batch soil slurry reactor (SBR) operated under anoxic–aerobic sequence. The results indicated that tetryl was co-metabolically converted to aniline under anoxic conditions with molasses as the growth substrate. The gas chromatographic/mass spectrometric analysis of the soil slurry showed various metabolites, identified as trinitrobenzeneamine, dintrobenzenediamine, nitroaniline and aniline. Aniline was not metabolized further under anoxic conditions. When the soil slurry reactor was operated under aerobic conditions, the aniline concentration was reduced to below the detection limit (0.05 ppm). This metabolic conversion of tetryl is probably of value in the treatment of tetryl-contaminated soil and ground water, such as those found at the Joliet army ammunition plant site in Illinois and the Iowa army ammunition plant site in Burlington, Iowa.     

Immobilisation of the D-2-haloacid dehalogenase fromPseudomonas putida strain AJ1/23

A variety of procedures were used to immobilise D-2-haloacid dehalogenase. Natural polymer supports were insufficiently robust to withstand degradation by high concentrations of 2-chloropropionate. The best results were obtained with enzyme covalently attached to controlled-pore glass via a diazo linkage. The immobilisation procedure was optimised with respect to enzyme loading, pH, temperature and the presence of substrate during attachment. Immobilisation significantly modified the kinetics of the enzyme, in particular improving its temperature stability and ability to withstand mildly alkaline conditions where it is most active. The performance of the immobilised preparation in batch and plug-flow bioreactors was assessed. Biocatalyst half-life in plug-flow reactors was better than in batch bioreactors whereas effectiveness factors, although concentration dependent in the batch reactor, were similar at least with 200 mM D,L-2-CPA as substrate.     

Continuous microbial desulfurization of coal--application of a multistage slurry reactor and analysis of the interactions of microbial and chemical kinetics

Microbial desulfurization of coal by pyrite oxidizing bacterial enrichment cultures has been studied in air-agitated slurry reactors of 4- and 20-L volumes. Batch experiments showed that inoculation with an active bacterial culture is essential to minimize the lag phase, although a considerable number of pyrite oxidizing bacteria was found on the coal prior to desulfurization. For detailed investigations of kinetics, energy requirements, and technical applicability, a bioreactor equipment consisting of a cascade of eight stages was developed and operated continuously. Microbial desulfurization of coal-monitored by measuring the axial profile of dissolved iron concentration, real and maximum oxygen consumption rates, and cell concentration-at pulp densities to 30% was performed over a period of 200 days without any disturbances concerning the aeration system, fluidization, transport of solids and microbial growth. At a pulp density of 20%, a pyrite conversion of 68% was achieved after the third reactor stage at a total residence time of five days in the first three stages. The kinetics of pyrite degradation were found to be well described by a rate equation of first order in pyrite surface area concentration if the pyrite is directly accessible for microbial attack. Rate constants were determined to 0.48 mg pyrite/(cm(2) day) in the first and to 0.24 mg pyrite/(cm(2) day) in the following reactor stages. Kinetic models taking into account adsorption/desorption as well as growth kinetics failed to describe the observed reaction rates. However, a model treating pyrite degradation and microbial growth kinetics formalistically seems to be applicable when backmixing between the reactor stages can be avoided. The advantage of a multistage reactor in comparison to single-stage equipment was shown by calculation. To obtain a pyrite conversion of 68%, a three-stage reactor would require only 58% of the volume of single-stage equipment.Measurement of oxygen consumption rates proved to provide quickly and easily measurable parameters to observe microbial coal desulfurization in technical scale: the real oxygen consumption rate is correlated to the pyrite oxidation rate and the maximum oxygen consumption rate is correlated to the concentration of viable cells. The Y(o/s) coefficient for the amount of oxygen consumed per mass unit of pyrite oxygen was determined to approximately 0.33 in comparison to 1.0 which can be calculated from stoichiornetry. This could yet not be explained. Chemical leaching experiments as well as sulfur analyses of desulfurized coal samples showed that the microorganisms play the main role in degradation of pyrite from coal and that pyrite oxidation by ferric iron can be neglected.     

Effect of aeration rate and waste load on evolution of volatile fatty acids and waste stabilization during thermophilic aerobic digestion of a model high strength agricultural waste

Thermophilic aerobic digestion (TAD) is a relatively new, dynamic and versatile low technology for the economic processing of high strength waste slurries. Waste so treated may be safely disposed of or reused. In this work a model high strength agricultural waste, potato peel, was subjected to TAD to study the effects of oxygen supply at 0.1, 0.25, 0.5 and 1.0 vvm (volume air per volume slurry per minute) under batch conditions at 55 degrees C for 156 h on the process. Process pH was controlled at 7.0 or left unregulated. Effects of waste load, as soluble chemical oxygen demand (COD), on TAD were studied at 4.0, 8.0, 12.0 and 16.0 gl(-1) (soluble COD) at pH 7.0, 0.5 vvm and 55 degrees C. Efficiency of treatment, as degradation of total solids, total suspended solids and soluble solid, as well as soluble COD significantly increased with aeration rate, while acetate production increased as the aeration rate decreased or waste load increased, signifying deterioration in treatment. Negligible acetate, and no other acids were produced at 1.0 vvm. Production of propionate and other acids increased after acetate concentration had started to decrease and, during unregulated reactions coincided with the drop in the pH of the slurry. Acetate production was more closely associated with periods of oxygen limitation than were other acids. Reduction in oxygen availability led to deterioration in treatment efficiency as did increase in waste load. These variables may be manipulated to control treated waste quality.