Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor

High-rate biological conversion of sulfide and nitrate in synthetic wastewater to, respectively, elemental sulfur (S⁰) and nitrogen-containing gas (such as N₂) was achieved in an expanded granular sludge bed (EGSB) reactor. A novel strategy was adopted to first cultivate mature granules using anaerobic sludge as seed sludge in sulfate-laden medium. The cultivated granules were then incubated in sulfide-laden medium to acclimate autotrophic denitrifiers. The incubated granules converted sulfide, nitrate, and acetate simultaneously in the same EGSB reactor to S⁰, N-containing gases and CO₂ at loading rates of 3.0 kg S m⁻³ d⁻¹, 1.45 kg N m⁻³ d⁻¹, and 2.77 kg Ac m⁻¹ d⁻¹, respectively, and was not inhibited by sulfide concentrations up to 800 mg l⁻¹. Effects of the C/N ratio on granule performance were identified. The granules cultivated in the sulfide-laden medium have Pseudomonas spp. and Azoarcus sp. presenting the heterotrophs and autotrophs that co-work in the high-rate EGSB-SDD (simultaneous desulfurization and denitrification) reactor.     

Biological nitrification–denitrification with alternating oxic and anoxic operations using aerobic granules

Efficient nitrification and denitrification of wastewater containing 1,700 mgl⁻¹ of ammonium-nitrogen was achieved using aerobic granular sludge cultivated at medium-to-high organic loading rates. The cultivated granules were tested in a sequencing batch reactor (SBR) fed with 6.4 or 10.2 kg NH₄⁺-N m⁻³ day⁻¹, a loading significantly higher than that reported in literature. With alternating 2 h oxic and 2 h anoxic operation (OA) modes, removal rate was 45.5 mg NH₄⁺-N g⁻¹ volatile suspended solids⁻¹ h⁻¹ at 6.4 kg NH₄⁺-N m⁻³ day⁻¹ loading and 41.3 ± 2.0 at 10.2 kg NH₄⁺-N m⁻³ day⁻¹ loading. Following the 60 days SBR test, granules were intact. The fluorescence in situ hybridization and confocal laser scanning microscopy results indicate that the SBR-OA granules have a distribution with nitrifers outside and heterotrophs outside that can effectively expose functional strains to surrounding substrates at high concentrations with minimal mass transfer limit. This microbial alignment combined with the smooth granule surface achieved nitrification–denitrification of wastewaters containing high-strength ammonium using aerobic granules. Conversely, the SBR continuous aeration mode yielded a distribution with nitrifers outside and heterotrophs inside with an unsatisfactory denitrification rate and floating granules as gas likely accumulated deep in the granules.     

Properties of phenol-removal aerobic granules during normal operation and shock loading

The physical structure and activity of aerobic granules, and the succession of bacterial community within aerobic granules under constant operational conditions and shock loading were investigated in one sequencing batch reactor over ten months. While the maturation phase of the granulation process began on day 30, the structure of microbial community changed markedly until after three months of reactor operation under constant conditions with a loading rate of 1.5 g phenol L⁻¹ day⁻¹. A shock loading of 6.0 g phenol L⁻¹ day⁻¹ from days 182–192 led to divergence of bacterial community, an inhibition of the biomass activity, and a decrease in phenol removal rate in the reactor. However, phenol was still completely removed under this disturbance. After the shock loading, the mean sizes of aerobic granules increased, and the activity of the microbial population within the granules decreased, although there appeared highly resilient for the dominant bacterial community of aerobic granules which mainly included β-Proteobacteria. Correlation analysis suggested that biomass concentration and biomass loading were significantly related to the community composition of aerobic granules during the whole operational period. The development of a relatively stable bacterial community in aerobic granules implied that those distinct dominant microbes in aerobic granules were favorably selected and proliferated under the operational conditions.     

Influence of aeration intensity on mature aerobic granules in sequencing batch reactor

Aeration intensity is well known as an important factor in the formation of aerobic granules. In this research, two identical lab-scale sequencing batch reactors with aeration intensity of 0.8 (R1) and 0.2 m³/h (R2) were operated to investigate the characteristics and kinetics of matured aerobic granules. Results showed that both aeration intensity conditions induced granulation, but they showed different effects on the characteristics of aerobic granules. Compared with the low aeration intensity (R2), the aerobic granules under the higher aeration intensity (R1) had better physical characteristics and settling ability. However, the observed biomass yield (Y _obs) in R1 [0.673 kg mixed liquor volatile suspended solids (MLVSS)/kg chemical oxygen demand (COD)] was lower than R2 (0.749 kg MLVSS/kg COD). In addition, the maximum specific COD removal rates (q _max) and apparent half rate constant (K) of mature aerobic granular sludge under the two aeration intensities were at a similar level. Therefore, the matured aerobic granule system does not require to be operated in a higher aeration intensity, which will reduce the energy consumption.     

Nitrifying granules cultivation in a sequencing batch reactor at a low organics-to-total nitrogen ratio in wastewater

It is possible to cultivate aerobic granular sludge at a low organic loading rate and organics-to-total nitrogen (COD/N) ratio in wastewater in the reactor with typical geometry (height/diameter = 2.1, superficial air velocity = 6 mm/s). The noted nitrification efficiency was very high (99%). At the highest applied ammonia load (0.3 ± 0.002 mg NH₄⁺–N g total suspended solids (TSS)⁻¹ day⁻¹, COD/N = 1), the dominating oxidized form of nitrogen was nitrite. Despite a constant aeration in the reactor, denitrification occurred in the structure of granules. Applied molecular techniques allowed the changes in the ammonia-oxidizing bacteria (AOB) community in granular sludge to be tracked. The major factor influencing AOB number and species composition was ammonia load. At the ammonia load of 0.3 ± 0.002 mg NH₄⁺–N g TSS⁻¹ day⁻¹, a highly diverse AOB community covering bacteria belonging to both the Nitrosospira and Nitrosomonas genera accounted for ca. 40% of the total bacteria in the biomass.     

Decolourization of anaerobically digested and polyaluminium chloride treated distillery spentwash in a fungal stirred tank aerobic reactor

Decolourization of anaerobically digested and polyaluminium chloride treated distillery spentwash was studied in a fungal stirred tank aerobic reactor without dilution of wastewater. Aspergillus niger isolate IITB-V8 was used as the fungal inoculum. The main objectives of the study were to optimize the stirrer speed for achieving maximum decolourization and to determine the kinetic parameters. A mathematical model was developed to describe the batch culture kinetics. Volumetric oxygen transfer coefficient (k _L a) was obtained using dynamic method. The maximum specific growth rate and growth yield of fungus were determined using Logistic equation and using Luedeking–Piret equation. 150 rpm was found to be optimum stirrer speed for overall decolourization of 87%. At the optimum stirrer speed, volumetric oxygen transfer coefficient (k _L a) was 0.4957 min⁻¹ and the maximum specific growth rate of fungus was 0.224 h⁻¹. The values of yield coefficient (Y _x/s) and maintenance coefficient (m _s) were found to be 0.48 g cells (g substrate)⁻¹ and 0.015 g substrate (g cells)⁻¹ h⁻¹.     

Microbial community in granules from a high-rate EGSB reactor

The spatial distribution, quantity and diversity of different microorganisms within anaerobic granular sludge from a lab-scale expanded granular sludge bed (EGSB) reactor operated at different organic loading rates were studied using florescent in situ hybridization (FISH), real time quantitative — polymerase chain reaction (RTQ-PCR) and denaturing gradient gel electrophoresis (DGGE) techniques. The results indicated that most Eubacteria were located in the outer layer of granule, while the Archaea which mainly were methanogens and more sensible to the environmental conditions were located in the inner layer of the granule. The quantity of Archaea was obviously less than that of Eubacteria in the granules, but increased with the increasing of organic loading rates of the reactor. As the organic loading rate of the reactor increased and the operating time elapsed, the Archaea community in the granules changed significantly. Seven typical DGGE bands were collected and sequenced, and found that the dominant species of Archaea in the granules operated in the last period were mainly Methanocorpusculum, Methanobacterium, Methanosaeta.     

Characteristics of aerobic granular sludge in a sequencing batch reactor with variable aeration

Aerobic granules can be used for the treatment of industrial or municipal wastewater, but high aeration rate is required for the stable operation of the granular sludge system. Therefore, the aim of this research was to reduce aeration rate greatly to decrease the energy consumption for the technology of aerobic granules. Based on the characteristics of sequencing batch reactor with distinct feast and famine periods, aeration rate was reduced from 1.66 to 0.55 cm s⁻¹ in the famine period after granules were formed. It was found that the settleability of aerobic granules in reactor R1 with reduced aeration was the same as that of aerobic granules in reactor R2 with constant aeration rate of 1.66 cm s⁻¹. However, the outer morphology of aerobic granules gradually changed from round shape to long shape, and minor population showed certain shift after aeration rate was reduced in the famine period. Since good settleability is the most essential feature of aerobic granules, it can be said that reducing aeration rate in famine period did not influence the stable operation of aerobic granular sludge system. Furthermore, the experimental results indicated that aeration rate in feast period was much more important to the stable operation of aerobic granules than that in famine period.     

Starvation is not a prerequisite for the formation of aerobic granules

Activated sludge with sludge volume index (SVI)₃₀ of 77 ml g⁻¹ and SVI₃₀ of 433 ml g⁻¹ was inoculated to start up reactors R1 and R2, respectively. In both R1 and R2, cycle time of 1 h and the influent chemical oxygen demand (COD) concentrations of 1,000 mg l⁻¹ were employed. Initial settling time of 2 min resulted in the loss of a substantial amount of biomass as wash-out and high effluent COD concentrations within the first week of operation. This implied that there was no starvation phase in each cycle of R1 and R2 during the first week of operation. However, aerobic granules with a size above 400 μm formed by day 7. Thus, it was concluded that starvation was not a prerequisite for the formation of aerobic granules. When cycle time was 1 h, the instability of aerobic granules was observed. When cycle time was prolonged to 1.5 h and granular sludge of 200 ml was used to start up reactor R3, the reactor R3 reached steady state within 1 week. SVI, size, and the morphology of granular sludge in R3 remained stable during the 47-day operation, which indicated that prolonged starvation time had positive effects on the stability of aerobic granules.     

Investigation on the formation and kinetics of glucose-fed aerobic granular sludge

Aerobic granular sludge was cultivated in a glass sequencing batch reactor (SBR) with glucose synthetic wastewater. The spherical shaped granules were observed on 4th day with the mean diameter of 0.1 mm. With the increase of chemical oxygen demand (COD) concentration of the influent, aerobic granules grew matured, the size of which ranged from 1.2 to 1.9 mm. The aerobic granular sludge could sustain high organic loading rate (about 4.0 g COD L⁻¹ d⁻¹), with good settling ability (settling velocity 36 m/h) and high biomass concentration (MLSS 6.7 ±0.2 g/L). Experimental data indicated that the substrate utilization and biomass growth kinetics followed Monod's kinetics model approximately. The corresponding kinetic coefficients of maximum specific substrate utilization rate (k), half velocity coefficient (K_s), growth yield coefficient (Y) and decay coefficient (K_d) were 13.2 d⁻¹, 275.8 mg/L, 0.183–0.250 mg MLSS/mg COD and 0.023–0.075 d⁻¹, respectively, which made aerobic granules have short setup period, high rate of substrate utilization and little surplus sludge.     

High-rate nitrogen removal from livestock manure digester liquor by combined partial nitritation–anammox process

In this study, combination of a partial nitritation reactor, using immobilized polyethylene glycol (PEG) gel carriers, and a continuous stirred granular anammox reactor was investigated for nitrogen removal from livestock manure digester liquor. Successful nitrite accumulation in the partial nitritation reactor was observed as the nitrite production rate reached 2.1 kg-N/m³/day under aerobic nitrogen loading rate of 3.8 kg-N/m³/day. Simultaneously, relatively high free ammonia concentrations (average 50 mg-NH₃/l) depressed the activity of nitrite oxidizing bacteria with nitrate concentration never exceeding 3% of TN concentration in the effluent of the partial nitritation reactor (maximum 35.2 mg/l). High nitrogen removal rates were achieved in the granular anammox reactor with the highest removal rate being 3.12 kg-N/m³/day under anaerobic nitrogen loading rate of 4.1 kg-N/m³/day. Recalcitrant organic compounds in the digester liquor did not impair anammox reaction and the SS accumulation in the granular anammox reactor was minimal. The results of this study demonstrated that partial nitritation–anammox combination has the potential to successfully remove nitrogen from livestock manure digester liquor.     

Potential cause of aerobic granular sludge breakdown at high organic loading rates

Aerobic sludge granules are compact, strong microbial aggregates that have excellent settling ability and capability to efficiently treat high-strength and toxic wastewaters. Aerobic granules disintegrate under high organic loading rates (OLR). This study cultivated aerobic granules using acetate as the sole carbon and energy source in three identical sequencing batch reactors operated under OLR of 9–21.3 kg chemical oxygen demand (COD) m⁻³ day⁻¹. The cultivated granules removed 94–96% of fed COD at OLR up to 9–19.5 kg COD m⁻³ day⁻¹, and disintegrated at OLR of 21.3 kg COD m⁻³ day⁻¹. Most tested isolates did not grow in the medium at >3,000 mg COD l⁻¹; additionally, these strains lost capability for auto-aggregation and protein or polysaccharide productivity. This critical COD regime correlates strongly with the OLR range in which granules started disintegrating. Reduced protein quantity secreted by isolates was associated with the noted poor granule integrity under high OLR. This work identified a potential cause of biological nature for aerobic granules breakdown.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Rates of intercalator-driven platination of DNA determined by a restriction enzyme cleavage inhibition assay

A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking platinum–acridine agents represented by the formula [Pt(am₂)LCl](NO₃)₂, where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU). The formation of monofunctional adducts in the target sequence 5′-CGA was studied in a 40-base-pair probe containing the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel electrophoresis. The formation of monoadducts is approximately 3 times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am₂ is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k _obs = 1.4 ± 0.37 × 10⁻⁴ s⁻¹ (t _1/2 = 83 ± 22 min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k _obs = 5.7 ± 0.58 × 10⁻⁴ s⁻¹, t _1/2 = 21 ± 2 min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity (compound 3, k _obs = 2.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 55 ± 10 min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k _obs = 1.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 104 ± 38 min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds. Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine–purine cross-links are discussed.     

Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study

The effect of individual environmental conditions (pH, pO₂, temperature, salinity, concentration of ethanol, propanol, tryptone and yeast extract) on the specific growth rate as well as ethanol and glycerol production rate of Saccharomyces cerevisiae S288C was mapped during the fermentative growth in aerobic auxo-accelerostat cultures. The obtained steady-state values of the glycerol to ethanol formation ratio (0.1 mol mol⁻¹) corresponding to those predicted from the stoichiometric model of fermentative yeast growth showed that the complete repression of respiration was obtained in auxostat culture and that the model is suitable for calculation of Y _ATP and Q _ATP values for the aerobic fermentative growth. Smooth decrease in the culture pH and dissolved oxygen concentration (pO₂) down to the critical values of 2.3 and 0.8%, respectively, resulted in decrease in growth yield (Y _ATP) and specific growth rate, however the specific ATP production rate (Q _ATP) stayed almost constant. Increase in the concentration of biomass (>0.8 g dwt l⁻¹), propanol (>2 g l⁻¹) or NaCl (>15 g l⁻¹) lead at first to the decrease in the specific growth rate and Q _ATP, while Y _ATP was affected only at higher concentrations. The observed decrease in Q _ATP was caused by indirect rather than direct inhibition of glycolysis. The increase in tryptone concentration resulted in an increase in the specific growth rate from 0.44 to 0.62 h⁻¹ and Y _ATP from 12.5 to 18.5 mol ATP g dwt⁻¹. This study demonstrates that the auxo-accelerostat method, besides being an efficient tool for obtaining the culture characteristics, provides also decent conditions for the experiments elucidating the control mechanisms of cell growth.     

Biosorption and biodegradation of pentachlorophenol (PCP) in an upflow anaerobic sludge blanket (UASB) reactor

In order to understand the fate of PCP in upflow anaerobic sludge blanket reactor (UASB) more completely, the sorption and biodegradation of pentachlorophenol (PCP) by anaerobic sludge granules were investigated. The anaerobic granular sludge degrading PCP was formed in UASB reactor, which was seeded with anaerobic sludge acclimated by chlorophenols. At the hydraulic retention time (HRT) of 20–22 h, and PCP loading rate of 200–220 mg l⁻¹ d⁻¹, UASB reactor exhibited good performance in treating wastewater which containing 170–180 mg l⁻¹ PCP and the PCP removal rate of 99.5% was achieved. Sequential appearance of tetra-, tri-, di-, and mono-chlorophenol was observed in the reactor effluent after 20 mg l⁻¹ PCP introduction. Sorption and desorption of PCP on the anaerobic sludge granules were all fitted to the Freundlich isotherm equation. Sorption of PCP was partly irreversible. The Freundlich equation could describe the behavior of PCP amount sorbed by granular sludge in anaerobic reactor reasonably well. The results demonstrated that the main mechanism leading to removal of PCP on anaerobic granular sludge was biodegradation, not sorption or volatization.     

Structure and stability of aerobic granules cultivated under different shear force in sequencing batch reactors

The cultivation of stable aerobic granules as well as granular structure and stability in sequencing batch reactors under different shear force were investigated in this study. Four column sequencing batch reactors (R1–R4) were operated under various shear force, in terms of superficial upflow air velocity of 0.8, 1.6, 2.4, and 3.2 cm s⁻¹, respectively. Aerobic granules were formed in all reactors in the experiment. It was found that the magnitude of shear force has an important impact on the granule stability. At shear force of 2.4 and 3.2 cm s⁻¹, granules can maintain a robust structure and have the potential of long-term operation. Granules developed in low shear force (R1, 0.8 cm s⁻¹ and R2, 1.6 cm s⁻¹) deteriorated to large-sized filamentous granules with irregular shape, loose structure and resulted in poor performance and operation instability. Granules cultivated under high shear force (R3, 2.4 cm s⁻¹ and R4, 3.2 cm s⁻¹) stabilized to clear outer morphology, dense and compact structure, and with good performance in 120 days operation. Fractal dimension (D_f) represents the internal structure of granules and can be used as an important indicator to describe the structure and stability of granules. Due to the combined effects of shear force and growth force, the mature granules developed in R3 and R4 also displayed certain differences in granular structure and characteristics.     

Tetrachloroethene dechlorination kinetics by Dehalospirillum multivorans immobilized in upflow anaerobic sludge blanket reactors

Tetrachloroethene (C₂Cl₄) dechlorination kinetics in upflow anaerobic sludge blanket (UASB) reactors was determined after introducing de novo activities into the granular sludge. These activities were introduced by immobilizing Dehalospirillum multivorans in a test reactor containing unsterile granular sludge, and in a reference reactor, R1, containing sterile granular sludge. A second reference reactor, R2, contained only unsterile granular sludge and served as a control. The kinetic experiments were performed by pulsing the reactors with C₂Cl₄ in a recirculating batch mode. Formate and acetate were added as electron donor and carbon source. Both reactors inoculated with D. multivorans dechlorinated C₂Cl₄ to an equimolar amount of C₂H₂Cl₂ with only traces of C₂HCl₃ in the effluent. In the control reactor, C₂HCl₃ accumulated before C₂H₂Cl₂ was produced. A computer simulation program (AQUASIM) was used to estimate the kinetic parameters. The half-saturation constants (K _s) for C₂Cl₄ and C₂HCl₃ were almost equal in the reactors containing D.␣multivorans (17 μM and 18 μM for C₂Cl₄; 26 μM and 28 μM for C₂HCl₃), indicating no influence of sludge bacteria on the affinity of D. multivorans for C₂Cl₄ and C₂HCl₃. The maximum dechlorination rates (k _m X _B) were about twice as high in the reactor containing D.␣multivorans immobilized in sterile sludge (11 mmol C₂Cl₄ l sludge⁻¹ day⁻¹ and 27 mmol C₂HCl₃ l sludge⁻¹ day⁻¹) than in the test reactor (4.4 mmol C₂Cl₄ l sludge⁻¹ day⁻¹ and 15 mmol C₂HCl₃ l sludge⁻¹ day⁻¹). Compared to other C₂Cl₄-degrading systems, the dechlorination rates of the inoculated reactors and their affinities for C₂Cl₄ and C₂HCl₃ were high. Therefore, introduction of de novo activity is promising for the use of anaerobic reactors to bioremediate C₂Cl₄-polluted water. Received: 5 November 1998 / Received revision: 25 January 1999 / Accepted: 31 January 1999     

Effects of hydrocarbon additions on gas-liquid mass transfer coefficients in biphasic bioreactors

The effects of aliphatic hydrocarbons (n-hexadecane andn-dodecane) on the volumetric oxygen mass transfer coefficient (k _L a) were studied in flat alveolar airlift reactor and continuous stirred tank reactors (CSTRs). In the flat alveolar airlift reactor, high aeration rates (>2 vvm) were required in order to obtain efficient organic-aqueous phase dispersion and reliablek _L a measurements. Addition of 1% (v/v)n-hexadecane orn-dodecane increased thek _l a 1.55-and 1.33-fold, respectively, compared to the control (superficial velocity: 25.8×10⁻³ m/s, sparger orifice diameter: 0.5 mm). Analysis of the gas-liquid interfacial areaa and the liquid film mass transfer coefficientk _L suggests that the observedk _L a increase was a function of the media's liquid film mass transfer. Addition of 1% (v/v)n-hexadecane orn-dodecane to analogous setups using CSTRs led to ak _L a increase by a factor of 1.68 and 1.36, respectively (superficial velocity: 2.1×10⁻³ m/s, stirring rate: 250 rpm). These results propose that low-concentration addition of oxygen-vectors to aerobic microbial cultures has additional benefit relative to incubation in purely aqueous media.     

Quantitative Structure-Activity Relationships for the Pre-Steady State of <Emphasis Type="Italic">Pseudomonas Species</Emphasis> Lipase Inhibitions by <Emphasis Type="Italic">p</Emphasis>-Nirophenyl-<Emphasis Type="Italic">N</Emphasis>-Substituted Carbamates

The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1–6) are composed of two steps: (1) formation of the non-covalent enzyme–inhibitor complex (E:I) from the inhibitor and the enzyme and (2) formation of the tetrahedral enzyme–inhibitor adduct (E–I) from the E:I complex. From a stopped-flow apparatus, the dissociation constant for the E:I complex, K_S, and the rate constant for formation of the tetrahedral E–I adduct from the E:I complex, k₂ are obtained from the non-linear least-squares of curve fittings of first-order rate constant (k_obs) versus inhibition concentration ([I]) plot against k_obs=k₂+k₂[I]/(K_S+[I]). Values of pK_S, and log k₂ are linearly correlated with the σ^* values with the ρ^* values of −2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to the ρ^* value of −2.0. The tetrahedral E–I adducts on the other hand are more negative charges than the E:I complexes due to the ρ^* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme.