Preliminary study of a suspended growth predenitrification system

A laboratory study has been conducted to obtained preliminary process information of a suspended growth Predenitrification (SGPDN)system. System performance was evaluated, in terms of chemical oxygen demand (COD) removal, NH(3)-N removal, system biomass yield and inventory, and effluent qualities, at different solids retention times (SRTs) and recycle ratios. Chemical oxygen demand removal in an SGPDN system occurs mainly in the anoxic reactor, which accounts for 94% of total COD removal. The overall COD removal rate is independent of recycle ratio (ranging from 2-5) used in this study; however, effluent COD increase with increasing recycle ratio. The observed anoxic and aerobic COD removal rates decrease with increasing SRT. The NH(3)-N removal in an SGPDN system is induced by two mechanisms: assimilatory NH(3)-N requirement for biomass production in the anoxic reactor and nitrification in the aerobic reactor. The observed anoxic NH(3)-N removal rate relates directly to the anoxic COD removal rate and agrees fairly well with the assimilatory NH(3)-N requirement theoretically predicted. The overall NH(3)-N removal rate is independent of SRTs and recycle ratios used in this study. Biomass yield in an SGPDN system occurs mainly in the anoxic reactor. However, uniform distribution of biomass throughout the entire system is obtained because of the high recycle rate used. The observed biomass yield (Y(O)) decreases with increasing STR. Tertiary treatment efficiency can be achieved in an SGPDN system. More than 90% reduction in feed COD., feed NH(3)-N, and NO(2) + NO(3)-N is obtained at all SRTs and recycle ratios used in this study. Higher MLVSS loading rates can be applied to a final clarifier without impairing its separation efficiency because of the excellent settleability of the Predenitrification activated sludge.     

Two‐compartment processing as a tool to boost recombinant protein production

Pichia pastoris is used extensively as a production platform for many recombinant proteins. The dissolved oxygen (DO) is one of the most important factors influencing protein production. The influence of the DO on productivity has not been studied independent from the feed rate. In this work, various DO levels were investigated independent from the feed rate. The model system was recombinant P. pastoris under the control of methanol‐induced alcohol oxidase promoter, which expressed HRP as the target protein. No significant effect was observed in terms of titer and specific productivity, which is a confirmation of the fact that the DO in a one‐compartment system cannot boost productivity for the model system under study. Hence, a two‐compartment system (a single reactor coupled with a plug flow reactor) was designed and implemented in order to apply oxygen‐related stress in the plug flow reactor and allow the cells to be recovered in the main reactor. Doing so, more than two‐fold increase in the titer and productivity and three‐fold increase in protein‐specific activity were achieved. Hence, partial application of oxygen‐related stress in the two‐compartment system was proposed as a process technology to enhance protein production.     

Analysis of biological wastewater treatment processes using multicomponent gas phase mass balancing.

A reactor system using off-gas analysis was developed for analyzing wastewater treatment process reactions. Using a mass spectrometer for the gas analysis provides the ability to simultaneously measure several gas components (such as oxygen, nitrogen, carbon dioxide, and argon). One of the benefits of the reactor design was the precise control of the dissolved oxygen concentration, uncoupled from the system turbulence, which was controlled via a gas recycle loop. This feature allowed control of the turbulence within the reactor without any need for mechanical stirring. Using oxygen as the test gas, the reactor was shown to perform well in the measurement of oxygen uptake rate of nitrifying activated sludge. The oxygen uptake rate calculations were made using a simple calibration method developed for the reactor system. The reactor was able to provide precise and accurate results for this test case. Furthermore, the system was capable of measuring under dynamic process conditions, as well as when the process rates were constant (steady state).     

Biomass recycling and species competition in continuous cultures

A chemostat with cell feedback is analyzed for three kinds of limiting nutrient: a substrate dissolved in the inflow, a gas bubbled directly into the reactor, and light. The effects of recycle are distinct in each case, because the relationships between hydraulic detention time and nutrient inflow are different for each type of nutrient, Effluent recycle, in which the recycle stream is more dilute than the reactor, is discussed in terms of cell detention time and nutrient limitation. Results from chemostat cultures of the blue-green alga, Spirulina geitleri, demonsrtat cell feedback under light limitation. Maximum Productivity is fixed by the incident light intensity. At a particular dilution rate recycling increases or decreases productivity by taking cell density closer or further from the optimum density. Cell recycle with heterogeneous populations can change the outcome of species competition. Selective recycling of one species can reverse this outcome or stabilize coexistence by its selective effect on cell detention time. Experimental results from light-limited mixed cultures of S. geitleri and a Chlorella sp. verify this.     

Effects of quantitative shock loadings on the constant recycle sludge concentration activated-sludge process

The reliability of the process of Ramanathan and Gaudy (Biotechnol Bioeng., 13 , 125 (1971)) for the completely mixed activated-sludge process holding the recycle cell concentration, X_R, as a system constant with respect to step changes in hydraulic retention time was investigated. The experiments were run at initial dilution rates of ?, ?, ¼, and ½ hr^?1 treating a soft drink bottling wastewater. The influent substrate concentration was maintained at 1000 mg/liter chemical oxygen demand and the hydraulic recycle ratio at 0.3. The recycle sludge concentration was maintained at about 7000 mg/liter. It was found that the system could accommodate hydraulic shock loads up to 200% positive changes and down to 50%negative changes without disruption of the effluent quality. Shorter retention time of the range studied, from 2 to 8 hr, has the advantage of shorter response time with respect to the response of the concentration of biological solids in the reactor.     

Bioremediation of selenite in oil refinery wastewater

Selenium-oxyanion-containing wastewater, with levels of selenite as high as 3690 g Se/l and very low levels of selenate, was treated in a laboratory-scale biological reactor system inoculated with the selenate-respiring bacterium Thauera selenatis. The wastewater contained selenite that had been removed from refinery effluent wastewater using iron-coprecipitation followed by selenite release to yield a more concentrated selenium-containing wastewater. The reactor system consisted of recycling sludge-blanket (500 ml; 200 g sand) and fluidized-bed reactors (500 ml; 150 g sand). The flow rate through the reactor system was 3.5 ml/min. The carbon source fed into the reactor was acetate (3 mM); nitrate was also present (3 mM). The selenium oxyanion levels in the wastewater were reduced by 95%. T. selenatis was the only selenate-reducing bacterium detected in the reactor system and it presumably reduced a portion of the selenate present in the water to selenite. The selenite present in the water, and that formed by selenate reduction, was reduced both by the Thauera and by a population of denitrifying bacteria also present in high numbers in the reactor system.     

Effect of organic substrates on biological sulphide oxidation

Summary Neither acetate nor higher fatty acids and glucose have a significant effect on the biotechnological process for sulphide removal at 20° C, in which sulphide is oxidized to sulphur using oxygen. The oxidation of acetate and propionate with oxygen is mainly dependent on the sulphide and oxygen concentrations in the reactor. The occurrence of Thiothrix filaments in sulphide-removing waste-water treatment systems has been investigated using a fixer-film upflow reactor. The influent of this reactor consisted of anaerobically treated paper-mill waste-water, with a sulphide concentration of 140 mg/1. It was found that sulphide loading rate is the decisive parameter as to whether or not Thiothrix will develop in a sulphide-removing reactor. Offprint requests to: C. J. N. Buisman     

Single-cell protein production from spent sulfite liquor utilizing cell-recycle and computer monitoring

Spent sulfite liquor (SSL), a waste product of the paper pulping industry, is produced at a rate of 1 ton (dry basis) per ton of pulp. The sugar content of SSL is about 30 g/L. To reduce the biological oxygen demand (BOD) of SSL before disposal, torula yeast (Candida utilis) is produced by a continuous culture process, the productivity of which is limited by sugar concentration and cell growth rate. To increase productivity, a recycle system has been designed and tested. Cells were sedimented continuously with a flocculating agent (bentonite) before being recycled to the fermentor. A bentonite concentration of 0.02 g/g cell was required. A computer monitoring system based on material balancing techniques was developed to monitor and control the recycle system. With this computer system, productivity was raised to 6.1 g/L h, with cell concentration up to 65 g/L in the recycle stream and 24 g/L in the fermentor. This represents a productivity increase of 150% over continuous culture with no recycle.     

Incorporating hydrodynamics into spatiotemporal metabolic models of bubble column gas fermentation

Gas fermentation has emerged as a technologically and economically attractive option for producing renewable fuels and chemicals from carbon monoxide (CO) rich waste streams. LanzaTech has developed a proprietary strain of the gas fermentating acetogen Clostridium autoethanogenum as a microbial platform for synthesizing ethanol, 2,3-butanediol, and other chemicals. Bubble column reactor technology is being developed for the large-scale production, motivating the investigation of multiphase reactor hydrodynamics. In this study, we combined hydrodynamics with a genome-scale reconstruction of C. autoethanogenum metabolism and multiphase convection–dispersion equations to compare the performance of bubble column reactors with and without liquid recycle. For both reactor configurations, hydrodynamics was predicted to diminish bubble column performance with respect to CO conversion, biomass production, and ethanol production when compared with bubble column models in which the gas phase was modeled as ideal plug flow plus axial dispersion. Liquid recycle was predicted to be advantageous by increasing CO conversion, biomass production, and ethanol and 2,3-butanediol production compared with the non-recycle reactor configuration. Parametric studies performed for the liquid recycle configuration with two-phase hydrodynamics showed that increased CO feed flow rates (more gas supply), smaller CO gas bubbles (more gas–liquid mass transfer), and shorter column heights (more gas per volume of liquid per time) favored ethanol production over acetate production. Our computational results demonstrate the power of combining cellular metabolic models and two-phase hydrodynamics for simulating and optimizing gas fermentation reactors.     

Phenol degradation in a three-phase biofilm fluidized sand bed reactor

A previous three phase fluidized sand bed reactor design was improved by adding a draft tube to improve fluidization and submerged effluent tubes for sand separation. The changes had little influence on the oxygen transfer coefficients(K _L a), but greatly reduced the aeration rate required for sand suspension. The resulting 12.5 dm³ reactor was operated with 1 h liquid residence time, 10.2dm³/min aeration rate, and 1.7–2.3 kg sand (0.25–0.35 mm diameter) for the degradation of phenol as sole carbon source. The K _La of 0.015 s^–1 gave more than adequate oxygen transfer to support rates of 180g phenol/h · m³ and 216 g oxygen/h · m³. The biomass-sand ratios of 20–35 mg volatiles/g gave estimated biomass concentrations of 3–6 g volatiles/dm³. Offline kinetic measurements showed weak inhibition kinetics with constants ofK _s=0.2 mg phenol/dm³, K _o2=0.5 mg oxygen/dm³ and KinI= 122.5 mg phenol/dm³. Very small biofilm diffusion effects were observed. Dynamic experiments demonstrated rapid response of dissolved oxygen to phenol changes below the inhibition level. Experimentally simulated continuous stagewise operation required three stages, each with 1 h residence time, for complete degradation of 300 mg phenol/dm³ · h.     

The application of constant recycle solids concentration in completely mixed oxygen activated sludge process

For better operational control of the completely mixed oxygen activated sludge process (CMOAS), a study concerning the kinetics, performance, and operational stability of the Ramanathan-Gaudy model was conducted. Short-term experiments were conducted at various dilution rates (1/9, 1/6, 1/3, 1/1.5, and 1/1.0 hr^?1) by using two recycle solids concentration values (5000 and 10,000 mg/liter). The influent substrate was an actual industrial organic wastewater (soft drink waste) and its concentration was maintained at 1000 mg/liter COD. The hydraulic recycle ratio, α, was maintained at 0.30. It was found that for CMOAS system with constant recycle cell concentration, a “steady state” with respect to reactor biological solids and effluent COD at different dilution rates could be attained. No appreciable dilute-out of reactor biological solids and substrate was observed up to the dilution rate of 1 hr^?1 for both systems of different X_R (5000 and 10,000 mg/liter). For the system of X_R = 5000 mg/liter, except the dilution rate of hr^?1, the effluent filtrate COD was lower than 100 mg/liter, the aerator biological solids concentration was about 1550 mg/liter, and the COD removal efficiency was higher than 90% for all dilution rates. For the system of X_R = 10,000 mg/liter, the effluent filtrate COD was lower than 71 mg/liter, the aerator biological solids concentration was about 2750 mg/liter, and the COD removal efficiency was higher than 90% throughout all the dilution rates selection in the present study. The value of the Sludge Volume Index (SVI) was the range of 37.0 to 58.5 and provided good settleability of sludge. The sludge yield was 0.53 for the system of X_R = 5000 mg/liter and 0.57 for the system of X_R = 10,000 mg/liter. The carbohydrate and the protein content of the cells were 10.1–21.6% and 35.6–50.6%, respectively. For predicting the reactor biological solid and effluent COD of the CMOAS system by using the Ramanathan-Gaudy model, two sets of values for the biological kinetic constants should be considered since it provided the best fit of predicted values of the observed values. In the present study, μ_m = 0.4 hr^?1, k_s = 92 mg/liter for 1/3 ? D ? 1, and μ_m = 0.05 hr^?1, k_s = 11.1 mg/liter for 1/9 ? D < 1/3 were used to calculate the predicted values of reactor biological solid and effluent filtrate COD.     

Wetland treatment of leachate from a closed landfill

A wetland system has operated seasonally at Saginaw Township, MI, USA, for ten years. The system consists of extraction, aeration, settling, intermittent vertical sand filtration, a surface flow wetland treatment with recycle, and discharge to the Tittibawassee River. The 0.85 ha cattail wetland treats the full leachate flow, with a total system detention time of 180 days. The high recycle rate creates a lesser wetland detention time of 60 days. Ammonia is the principal contaminant of concern, because it occurs at high concentrations, typically 300–500 mg/L. Ammonia mass reduction averaged 99.5% for the last nine years, with a 95% mass removal in the startup year. Metals were not present in all samples, with modest reductions in those always present (zinc 16%, arsenic 29%, barium 78%, chromium 67%). Volatile organic compounds were removed to below detection, excepting BTEX, which occurred in only 2% of the outflow samples. Base neutral organics, PCBs and pesticides were also removed to below detection, excepting phthalates with an outlet detection frequency of 29%. No pesticides or PCBs were detected in the system outflow. The ammonia removal rate coefficients for the wetland (12 m/yr) was at the 55th percentile of the distribution for other surface flow wetlands. The vertical filter was likely oxygen limited, and functioned with an apparent oxygen utilization of 30 gO/(m² d).     

Characterization of a modified rotating disk reactor for the cultivation of Staphylococcus epidermidis biofilm

Aims: The purpose of this study was to develop a system that would allow biofilms to be cultivated under strictly defined conditions in terms of dissolved oxygen, fluid shear and to assess whether the method was suitable for the detection of respiratory activity stratification in biofilm samples. Methods: The system is a modified version a commercially available laboratory biofilm reactor and incorporates a number of features such as the provision of defined levels of dissolved oxygen, constant average shear, enhanced gas–liquid mass transfer, aseptic operation and the ability to remove biofilm for ex situ analysis during or after continuous cultivation. Conclusions: The system was shown to be effective for the characterization of the effects of dissolved oxygen on a pure culture of Staphylococcus epidermidis. The versatility of the system offers the potential for cultivating pure culture biofilm in defined, controlled conditions and facilitates a range of analyses that can be performed ex situ. Significance and Impact of the Study: The ability to provide strict regulation of environmental conditions and enhanced transfer of oxygen to the biofilm during cultivation are important, first because oxygen is known to regulate biofilm development in several micro‐organisms and second because many conventional biofilm cultivation systems may not provide adequate oxygen supply to the biofilm.     

Characteristics of unbuffered gel-immobilized urease particles. II. Overall rate of reaction

The overall rates of reaction of unbuffered gel-immobilized urease particles have been investigated with the aid of a packed-bed differential recycle reactor. Both substrate and enzyme concentrations have received attention. Cylindrical gel particles contained within impermeable tubelets were used to provide the physical strength necessary for the packed-bed arrangement and a one dimensional diffusion path to aid understanding of the complex interactions between substrate and product diffusion, and their effect on the reactions taking place. The experimental data have been interpreted with the aid of an enzyme rate equation (ERE) which relates the free solution characteristics of the enzyme to the conditions within a diffusion limited particle. The internal hydrogen ion profiles have been accommodated by a lumped parameter, the apparent pH (pH_app). Two methods have been suggested for the calculation of pH_app and the loss of activity on particle preparation, these methods are based on the use of the ERE in conjunction with experimental data.     

Oxygen transfer characteristics of miniaturized bioreactor systems

Since their introduction in 2001 miniaturized bioreactor systems have made great advances in function and performance. In this article the dissolved oxygen (DO) transfer performance of submilliliter microbioreactors, and 1–10 mL minibioreactors was examined. Microbioreactors have reached k_La values of 460 h^?1, and are offering instrumentation and some functionality comparable to production systems, but at high throughput screening volumes. Minibioreactors, aside from one 1,440 h^?1 k_La system, have not offered as high rates of DO transfer, but have demonstrated superior integration with automated fluid handling systems. Microbioreactors have been typically limited to studies with E. coli, while minibioreactors have offered greater versatility in this regard. Further, mathematical relationships confirming the applicability of k_La measurements across all scales have been derived, and alternatives to fluorescence lifetime DO sensors have been evaluated. Finally, the influence on reactor performance of oxygen uptake rate (OUR), and the possibility of its real‐time measurement have been explored. Biotechnol. Bioeng. 2013; 110: 1005–1019. © 2012 Wiley Periodicals, Inc.     

Physiological state of a microbial community in a biomass recycle reactor

The transition in physiological state was investigated between a carbon-limited chemostat population and microbes growing very slowly in a biomass recycle reactor. The mixed microbial population was metabolizing a mixture of biopolymers and linear alkylbenzene sulfonate, formulated to represent the organic load in graywater. Biomass increased 30-fold during the first 14 days after a shift from chemostat to biomass recycle mode. The ratios of ATP and RNA to cell protein decreased over the first days but then remained constant. The specific rate of CO₂ production by microbes in the reactor decreased 6-fold within 24 h after the shift, and respiratory potentials declined 2–3 fold during the first 7 days. Whereas chemostat cultures used equal proportions of organic carbon substrate for catabolism and anabolism, the proportion of organic substrate oxidized to CO₂ rose from 62 to 82% over the first 8 days in a biomass recycle reactor, and eventually reached 100% as this reactor population exhibited no net growth. Biomass recycle populations removed from the system and subjected to a nutritional shift-up did not immediately initiate exponential growth. The physiological state of cells in the biomass recycle reactor may be distinct from those grown in batch or continuous culture, or from starved cells. Received 02 June 1997/ Accepted in revised form 20 February 1998     

Deactivation studies of immobilized glucose oxidase

Studies have been performed in a tubular flow reactor to characterize the deactivation of immobilized glucose oxidase. The effects of oxygen concentration in the range of 0.09 to 0.467mM and hydrogen peroxide concentrations in the range of 0.1 to 10mM were studied. A simple mathematical model assuming first-order reaction and deactivation was found to describe the deactivation behavior adequately. The deactivation rate constant was found to increase with increasing levels of feed oxygen. Hydrogen peroxide was found to deactivate the enzyme severely and the deactivation rate constants were higher than those for oxygen deactivation. The influence of external and internal diffusion effects on the deactivation rate constant were examined. Although diffusional restrictions were negligible for oxygen transfer to the pellet, they were significant for transfer of hydrogen peroxide to the bulk stream. Increasing deactivation rates. Severe internal diffusion limitations were observed for the glucose oxidase system. However, for particle sizes in the range of 500 to 2000 μm, no effect on the rate of deactivation of the enzyme was observed.     

Design and evaluation of a continuous semipartition bioreactor for in situ liquid‐liquid extractive fermentation

Extractive fermentation (or in situ product removal (ISPR)) is an operational method used to combat product inhibition in fermentations. To achieve ISPR, different separation techniques, modes of operation and physical reactor configurations have been proposed. However, the relative paucity of industrial application necessitates continued investigation into reactor systems. This article outlines a bioreactor designed to facilitate in situ product extraction and recovery, through adapting the reaction volume to include a settler and solvent extraction and recycle section. This semipartition bioreactor is proposed as a new mode of operation for continuous liquid‐liquid extractive fermentation. The design is demonstrated as a modified bench‐top fermentation vessel, initially analysed in terms of fluid dynamic studies, in a model two‐liquid phase system. A continuous abiotic simulation of lactic acid (LA) fermentation is then demonstrated. The results show that mixing in the main reaction vessel is unaffected by the inserted settling zone, and that the size of the settling tube effects the maximum volumetric removal rate. In these tests the largest settling tube gave a potential continuous volumetric removal rate of 7.63 ml/min; sufficiently large to allow for continuous product extraction even in a highly productive fermentation. To demonstrate the applicability of the developed reactor, an abiotic simulation of a LA fermentation was performed. LA was added to reactor continuously at a rate of 33ml/h, while continuous in situ extraction removed the LA using 15% trioctylamine in oleyl alcohol. The reactor showed stable LA concentration of 1 g/L, with the balance of the LA successfully extracted and recovered using back extraction. This study demonstrates a potentially useful physical configuration for continuous in situ extraction.     

Continuous recombinant bacterial fermentations utilizing selective flocculation and recycle.

Selective recycle has successfully been used to maintain an unstable plasmid-bearing bacterial strain as dominant in a continuous reactor, whereas the culture reverts to 100% segregant cells when selective recycle is not used. The plasmid-bearing strain is slower growing and flocculent; however, when the cells lose their plasmid, the resulting segregant cells are nonflocculent and grow at a faster rate due to their decreased metabolic burden. Both types of cells exit a chemostat and enter an inclined settler where the flocculent plasmid-bearing cells are separated from the nonflocculent segregant cells by differential sedimentation. The underflow from the cell separator, which is enriched with plasmid-bearing cells, is recycled back to the chemostat, while the segregant cells are withdrawn off the top of the settler and discarded. The experimental results agree well with selective recycle reactor theory. On the basis of the theory, a criterion is presented that has been shown to successfully predict whether or not a selective recycle reactor can maintain a plasmid-bearing strain.