Impact of initial conditions on extant microbial kinetic parameter estimates: application to chlorinated ethene dehalorespiration

Monod kinetics are the foundation of mathematical models of many environmentally important biological processes, including the dehalorespiration of chlorinated ethene groundwater contaminants. The Monod parameters—q _max, the maximum specific substrate utilization rate, and K _S, the half-saturation constant—are typically estimated in batch assays, which are superficially simple to prepare and maintain. However, if initial conditions in batch assays are not chosen carefully, it is unlikely that the estimated parameter values will be meaningful because they do not reflect microbial activity in the environmental system of interest, and/or they are not mathematically identifiable. The estimation of q _max and K _S values that are highly correlated undoubtedly contributes significantly to the wide range in reported parameter values and may undermine efforts to use mathematical models to demonstrate the occurrence of natural attenuation or predict the performance of engineered bioremediation approaches. In this study, a series of experimental and theoretical batch kinetic assays were conducted using the tetrachloroethene-respirer Desulfuromonas michiganensis to systematically evaluate the effects of initial batch assay conditions, expressed as the initial substrate (S ₀)-to-initial biomass concentration (X ₀) ratio (S ₀/X ₀) and the S ₀/K _S ratio on parameter correlation. An iterative approach to obtain meaningful Monod parameter estimates was developed and validated using three different strains and can be broadly applied to a range of other substrates and populations. While the S ₀/X ₀ ratio is critical to obtaining kinetic parameter estimates that reflect in situ microbial activity, this study shows that optimization of the S ₀/K _S ratio is key to minimizing Monod parameter correlation.     

Studies on the relationship between specific growth rate and concentration of nitrogen source for heterogeneous microbial populations of sewage origin

Batch experiments were run using heterogeneous populations to determine whether a hyperbolic equation of the type suggested by Monod could be used to depict the relation between specific growth rate, μ, and NH₃-N concentration when ammonia N was the growth-limiting nutrient. The heterogeneous populations employed were developed from sewage seed grown on glucose at various levels of nitrogen and various dilution rates in completely mixed continuous flow reactors. It was found that the hyperbolic function could be used. Values of μ_m in the range of 0.4–0.7 hr^?1 were observed, and values of K_s, in general, ranged from 1.5 to 4.0 mg/l. Variation in the values of these growth “constants” did not follow any discernible pattern related to past growth history (i.e., COD:N ratio or dilution rate at which the cells were previously grown).     

Methanosarcinaceae and Acetate-Oxidizing Pathways Dominate in High-Rate Thermophilic Anaerobic Digestion of Waste-Activated Sludge

This study investigated the process of high-rate, high-temperature methanogenesis to enable very-high-volume loading during anaerobic digestion of waste-activated sludge. Reducing the hydraulic retention time (HRT) from 15 to 20 days in mesophilic digestion down to 3 days was achievable at a thermophilic temperature (55°C) with stable digester performance and methanogenic activity. A volatile solids (VS) destruction efficiency of 33 to 35% was achieved on waste-activated sludge, comparable to that obtained via mesophilic processes with low organic acid levels (<200 mg/liter chemical oxygen demand [COD]). Methane yield (VS basis) was 150 to 180 liters of CH₄/kg of VS_added. According to 16S rRNA pyrotag sequencing and fluorescence in situ hybridization (FISH), the methanogenic community was dominated by members of the Methanosarcinaceae, which have a high level of metabolic capability, including acetoclastic and hydrogenotrophic methanogenesis. Loss of function at an HRT of 2 days was accompanied by a loss of the methanogens, according to pyrotag sequencing. The two acetate conversion pathways, namely, acetoclastic methanogenesis and syntrophic acetate oxidation, were quantified by stable carbon isotope ratio mass spectrometry. The results showed that the majority of methane was generated by nonacetoclastic pathways, both in the reactors and in off-line batch tests, confirming that syntrophic acetate oxidation is a key pathway at elevated temperatures. The proportion of methane due to acetate cleavage increased later in the batch, and it is likely that stable oxidation in the continuous reactor was maintained by application of the consistently low retention time.     

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.     

Kinetic behavior of heterogeneous populations in completely mixed reactors

The kinetic behavior of heterogeneous microbial populations was studied in a continuous flow completely mixed reactor operated at various dilution rates. Glucose was used as the growth-limiting nutrient. The physiological growth parameters for cells harvested from continuous flow reactors were determined using batch experiments. It, was found that the growth parameters, maximum growth rate (μ_m), saturation constant (k_s), and cell yield (Y) vary for each dilution rate, and cannot be considered as precise constants in depicting the kinetic behavior of heterogeneous populations. In addition, it was found that the yield coefficients obtained from batch experiments were always lower than those obtained from continuous flow experiments. Levels of substrate and biological solids calculated for different dilution rates using growth constants from batch experiments did not agree with the experimental values observed in steady-state experiments. However, when the yield values from, the continuous flow experiments were used in conjunction with batch values for μ_m and k_s the theoretical and experimental dilute-out curves agreed fairly closely (within the range needed for engineering prediction) until the culture began to wash out of the unit. In general, the data substantiated the use of the single phase relationship between growth rate and substrate concentration described by the Monod equation, μ = μ_mS/(k_s + s).     

Acetate Degradation at 70°C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70°C

Anaerobic acetate degradation at 70°C and at 55°C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70°C and less than 15 days was needed at 55°C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55°C up to 90% of the COD was removed. Batch assays showed that sludges from two 70°C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70°C. Both 70°C sludges, as well as the 55°C sludge, produced methane at temperatures of 37 to 73°C. The 55°C sludge exhibited shorter lag phases than the 70°C sludges and higher specific methane production rates between 37 and 65°C.     

Growth ofPhormidium sp. in aerobic secondary piggery waste-water

The potential of a cyanobacteriumPhormidium sp., for the tertiary treatment of piggery waste-water chemical oxygen demand , 3000 mg·1^–1, using an aerobically stabilized secondary effluent, was studied. Batch cultures were carried out in 2-1 glass reactors and in a 30-1 glass-fibre carousel reactor. The nutrient removal efficiency as well as biomass production were compared in synthetic mineral medium and in different concentrations of aerobically stabilized piggery effluent. The best performance ofPhormidium sp. ocurred in diluted stabilized secondary effluent (1:1). Removal efficiencies were 100% for P-PO₄ ^–3, 50% for N-NH₄ ^–1 and 35% for N-NO₃ ^– in small-volume cultures; and 31% for P-PO₄ ^–3, 100% for N-NH₄ ^– and 70% for N-NO₃ ^– in an open carousel reactor. Biomass production on the aerobically stabilized effluent was six times higher in the 2-1 reactors and 1.7 times higher in the carousel reactor when compared to the synthetic medium.     

Cultivation of thermophilic methanogen KN-15 on H2-CO2 under pressurized conditions

Cultivation of the thermophilic methanogen KN-15 was carried out under pressurized batch and continuous conditions. In pressurized batch culture, both the turning point at which cell growth changed from exponential to linear and the growth rate during the linear growth phase increased with the rise of total pressure of the gas phase in the fermentor. The cell concentration reached 18.5 g dry cell/l after 10 h of batch cultivation under 3.0 × 10⁵ Pa pressurized conditions. Under pressurized continuous conditions, it was also observed that the cell concentration and cell productivities increased with the rise of total pressure of the gas phase. Cell and methane productivities of 3.0 g dry cell/l/h and 1.28 mol/l/h, respectively, were achieved in 3.0 × 10⁵ Pa pressurized continuous culture. According to the results from the Monod model application, the achievable cell productivities (V_max) and the Monod type saturation constant for cell productivities (K_s^′) were 12.8 g dry cell/l/h and 9.7 × 10⁵ Pa, respectively.     

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.     

Optimal design for CSTR's in series using reversible Michaelis-Menten reactions

An analytical expression is derived for the optimal design of a series of CSTR's performing reversible Michaelis-Menten kinetics in the liquid phase. The optimal design is based on minimum overall volume ofN reactors in series required to achieve a certain degree of substrate conversion. The reversible Michaelis-Menten equation is also able to explain competitive product inhibition and irreversible Michaelis-Menten kinetics. The reversible Michaelis-Menten kinetics covers three types of enzymatic reactions depending on the values of the rate constant for the forward (k _s) and reverse (k _p) reactions. An optimum design is obtained in the three cases ofK _s=K_p, K_s>K_p andK _s<K_p. The minimum overall reactors volume is compared with the more convenient equal-sized CSTR's. The effect of enzyme deactivation on the minimum overall reactors volume is investigated. The performance of a series of CSTR's is compared with plug-flow reactor. Glucose isomerization which exhibits reversible Michaelis-Menten kinetics is used as a model system for optimization.     

Biogas production from potato-juice, a by-product from potato-starch processing, in upflow anaerobic sludge blanket (UASB) and expanded granular sludge bed (EGSB) reactors

In this study, the utilization of potato-juice, the organic by-product from potato-starch processing, for biogas production was investigated in batch assay and in high rate anaerobic reactors. The maximum methane potential of the potato-juice determined by batch assay was 470 mL-CH₄/gVS-added. Anaerobic digestion of potato-juice in an EGSB reactor could obtain a methane yield of 380 mL-CH₄/gVS-added at the organic loading rate of 3.2 gCOD/(L-reactor.d). In a UASB reactor, higher organic loading rate of 5.1 gCOD/(L-reactor.d) could be tolerated, however, it resulted in a lower methane yield of 240 mL-CH₄/gVS-added. The treatment of reactor effluent was also investigated. By acidification with sulfuric acid to pH lower than 5, almost 100% of the ammonia content in the effluent could be retained during the successive up-concentration process step. The reactor effluent could be up-concentrated by evaporation to minimize its volume, and later be utilized as fertilizer.     

Properties of glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors

Glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the V_max value. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26s⁻¹ (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s⁻¹ (as a tetramer), the recycling rate of the active NAD moiety is 0.54 s⁻¹, and the apparent K_m value for glucose is 24 mM. Kinetically, lactate dehydrogenase behaves like a substrate with an apparent K_m value of 1.8 units·ml⁻¹ in this coupled reaction system with low coenzyme concentration. l-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size.     

Aerobic Mineralization of 2,6-Dichlorophenol by Ralstonia sp. Strain RK1

A new aerobic bacterium was isolated from the sediment of a freshwater pond close to a contaminated site at Amponville (France). It was enriched in a fixed-bed reactor fed with 2,6-dichlorophenol (2,6-DCP) as the sole carbon and energy source at pH 7.5 and room temperature. The degradation of 2,6-DCP followed Monod kinetics at low initial concentrations. At concentrations above 300 μM (50 mg · liter⁻¹), 2,6-DCP increasingly inhibited its own degradation. The base sequence of the 16S ribosomal DNA allowed us to assign the bacterium to the genus Ralstonia (formerly Alcaligenes). The substrate spectrum of the bacterium includes toluene, benzene, chlorobenzene, phenol, and all four ortho- and para-substituted mono- and dichlorophenol isomers. Substituents other than chlorine prevented degradation. The capacity to degrade 2,6-DCP was examined in two fixed-bed reactors. The microbial population grew on and completely mineralized 2,6-DCP at 2,6-DCP concentrations up to 740 μM in continuous reactor culture supplied with H₂O₂ as an oxygen source. Lack of peroxide completely stopped further degradation of 2,6-DCP. Lowering the acid-neutralizing capacity of the medium to 1/10th the original capacity led to a decrease in the pH of the effluent from 7 to 6 and to a significant reduction in the degradation activity. A second fixed-bed reactor successfully removed low chlorophenol concentrations (20 to 26 μM) with hydraulic residence times of 8 to 30 min.     

TEMPERATURE EFFECTS ON SILICON LIMITED GROWTH OF THE LAKE MICHIGAN DIATOM STEPHANODISCUS MINUTUS (BACILLARIOPHYCEAE)1

The effect of temperature on the silicon limited growth and nutrient kinetics of Stephanodiscus minutus Grun. was examined using batch and semicontinuous culture methods. Short-term batch culture methods gave maximum growth rates which were essentially constant over the temperature range of 10° to 20°C (μ₃= 0.71–0.80 d^?1). The half-saturation constant for growth (K_s) was significantly lowest at 10°C (K_s= 0.31 μM Si; 0.22–0.41), and higher at both 15°C (K_s= 1.03 μM Si; 0.68–1.47) and 20°C (K_s= 0.88 μM Si; 0.60–1.22). Two methods were used to evaluate the semicontinuous experiments. The Droop relationship showed that the minimum cell quota was about 1.50 × 10^?7 nmol Si cell^?1, but there was much overlap in the results at all three temperatures. The Monod growth relationship for the semicontinuous experiments gave estimates of K_s which were lowest at 15°C (K_s= 0.12 μM Si), and higher at 10°C (K_s= 0.68 μM Si) and 20°C (K_s= 1.24 μM Si), although 95% confidence intervals overlapped. The maximum growth rate estimates for the semicontinuous experiments were similar at 10° and 15°, and higher at 20°C, but the number of points used in making the calculations makes the results less reliable than those from batch cultures. Generally, there were no consistent significant differences in the silicon limited growth of S. minutus over the temperature range studied. Our values of K_s for S. minutus are the lowest recorded for a freshwater diatom, and are consistent with the distribution of this species in nature. Generally, this species becomes abundant in areas with high phosphorus loading and very low silicon levels (low Si:P loading rates). Stephanodiscus species are also fossil indicators of eutrophication in north temperate lakes.     

Influence of dissolved oxygen on the nitrification kinetics in a circulating bed biofilm reactor

The influence of dissolved oxygen concentration on the nitrification kinetics was studied in the circulating bed reactor (CBR). The study was partly performed at laboratory scale with synthetic water, and partly at pilot scale with secondary effluent as feed water. The nitrification kinetics of the laboratory CBR as a function of the oxygen concentration can be described according to the half order and zero order rate equations of the diffusion-reaction model applied to porous catalysts. When oxygen was the rate limiting substrate, the nitrification rate was close to a half order function of the oxygen concentration. The average oxygen diffusion coefficient estimated by fitting the diffusion-reaction model to the experimental results was around 66% of the respective value in water. The experimental results showed that either the ammonia or the oxygen concentration could be limiting for the nitrification kinetics. The latter occurred for an oxygen to ammonia concentration ratio below 1.5–2 gO₂/gN-NH₄ ⁺ for both laboratory and pilot scale reactors. The volumetric oxygen mass transfer coefficient (k _L a) determined in the laboratory scale reactor was 0.017?s^?1 for a superficial air velocity of 0.02?m s^?1, and the one determined in the pilot scale reactor was 0.040?s^?1 for a superficial air velocity of 0.031?m?s^?1. The k _L a for the pilot scale reactor did not change significantly after biofilm development, compared to the value measured without biofilm.     

Detoxification and partial mineralization of the azo dye mordant orange 1 in a continuous upflow anaerobic sludge-blanket reactor

In batch toxicity assays, azo dye compounds were found to be many times more toxic than their cleavage products (aromatic amines) towards methanogenic activity in anaerobic granular sludge. Considering the ability of anaerobic microorganisms to reduce azo groups, detoxification of azo compounds towards methanogens can be expected to occur during anaerobic wastewater treatment. In order to test this hypothesis, the anaerobic degradation of one azo dye compound, Mordant orange 1 (MO1), by granular sludge was investigated in three separate continuous upflow anaerobic sludge-blanket reactors. One reactor, receiving no cosubstrate, failed after 50 days presumably because of a lack of reducing equivalents. However, the two reactors receiving either glucose or a volatile fatty acids (acetate, propionate, butyrate) mixture, could eliminate the dye during operation for 217 days. The azo dye was reductively cleaved to less toxic aromatic amines (1,4-phenylenediamine and 5-aminosalicylic acid) making the treatment of MO1 feasible at influent concentrations that were over 25 times higher than their 50% inhibitory concentrations. In the reactor receiving glucose as cosubstrate, 5-aminosalicylic acid could only be detected at trace levels in the effluent after day 189 of operation. Batch biodegradability assays with the sludge sampled from this reactor confirmed the mineralization of 5-aminosalicylic acid to methane. Received: 11 July 1996 / Received revision: 18 September 1996 / Accepted: 18 September 1996     

Kinetics of Bacterial Growth on Chlorinated Aliphatic Compounds

With the pure bacterial cultures Ancylobacter aquaticus AD20 and AD25, Xanthobacter autotrophicus GJ10, and Pseudomonas sp. strain AD1, Monod kinetics was observed during growth in chemostat cultures on 1,2-dichloroethane (AD20, AD25, and GJ10), 2-chloroethanol (AD20 and GJ10), and 1,3-dichloro-2-propanol (AD1). Both the Michaelis-Menten constants (K_m) of the first catabolic (dehalogenating) enzyme and the Monod half-saturation constants (K_s) followed the order 2-chloroethanol, 1,3-dichloro-2-propanol, epichlorohydrin, and 1,2-dichloroethane. The K_s values of strains GJ10, AD20, and AD25 for 1,2-dichloroethane were 260, 222, and 24 μM, respectively. The low K_s value of strain AD25 was correlated with a higher haloalkane dehalogenase content of this bacterium. The growth rates of strains AD20 and GJ10 in continuous cultures on 1,2-dichloroethane were higher than the rates predicted from the kinetics of the haloalkane dehalogenase and the concentration of the enzyme in the cells. The results indicate that the efficiency of chlorinated compound removal is indeed influenced by the kinetic properties and cellular content of the first catabolic enzyme. The cell envelope did not seem to act as a barrier for permeation of 1,2-dichloroethane.     

Anaerobic treatment of distillery wastewater from barley-Shochu making by UASB

Distillery wastewater from barley-shochu making was anaerobically treated by a single upflow anaerobic sludge blanket (UASB), and stable operation at a volumetric TOC loading rate of 3 g/l·d could be attained by diluting raw wastewater to give a TOC concentration of less than 6,000 mg/l (18,000 mg/l as CODer). When the distribution of the concentrations of aliphatic acids at various levels within the reactor was investigated, it was found that the methanogenic gasification reaction did not occur in the sludge blanket of the reactor. Distillery wastewater from barley-shochu making with various TOC concentrations was preliminary adjusted to pH 7 and treated by a single passage without circulation of effluent to the bottom of the reactor. As a result, a maximum TOC removal rate (V_max) of 28.5 g/l·d and a saturation constant (K_s) of 0.63 g/l were obtained.     

Next-Generation Pyrosequencing Analysis of Microbial Biofilm Communities on Granular Activated Carbon in Treatment of Oil Sands Process-Affected Water

The development of biodegradation treatment processes for oil sands process-affected water (OSPW) has been progressing in recent years with the promising potential of biofilm reactors. Previously, the granular activated carbon (GAC) biofilm process was successfully employed for treatment of a large variety of recalcitrant organic compounds in domestic and industrial wastewaters. In this study, GAC biofilm microbial development and degradation efficiency were investigated for OSPW treatment by monitoring the biofilm growth on the GAC surface in raw and ozonated OSPW in batch bioreactors. The GAC biofilm community was characterized using a next-generation 16S rRNA gene pyrosequencing technique that revealed that the phylum Proteobacteria was dominant in both OSPW and biofilms, with further in-depth analysis showing higher abundances of Alpha- and Gammaproteobacteria sequences. Interestingly, many known polyaromatic hydrocarbon degraders, namely, Burkholderiales, Pseudomonadales, Bdellovibrionales, and Sphingomonadales, were observed in the GAC biofilm. Ozonation decreased the microbial diversity in planktonic OSPW but increased the microbial diversity in the GAC biofilms. Quantitative real-time PCR revealed similar bacterial gene copy numbers (>10⁹ gene copies/g of GAC) for both raw and ozonated OSPW GAC biofilms. The observed rates of removal of naphthenic acids (NAs) over the 2-day experiments for the GAC biofilm treatments of raw and ozonated OSPW were 31% and 66%, respectively. Overall, a relatively low ozone dose (30 mg of O₃/liter utilized) combined with GAC biofilm treatment significantly increased NA removal rates. The treatment of OSPW in bioreactors using GAC biofilms is a promising technology for the reduction of recalcitrant OSPW organic compounds.