Evaluation of steady-state-biofilm kinetics

Laboratory-scale biofilm reactors were used to evaluate a model of the kinetics of steady-state biofilm and the concept that there is a minimum concentration, S_min, below which no steady-state activity can occur. With acetate as the ratelimiting substrate, the steady-state concept of S_min was verified for naturally grown biofilms. Substrate removal and biofilm thickness declined rapidly as the substrate concentration approached S_min, which was 0.66 mg/liter for acetate. Using independently derived kinetic parameters, the model of steady-state-biofilm kinetics successfully predicted substrate utilization and biofilm thickness without the need for fitting factors. The results imply that organic materials may persist in water and wastewater, in part, because they are too low in concentration to supply sufficient energy to sustain the microorganisms.     

Film analysis of activated sludge microbial discs by the Taguchi method and grey relational analysis

A biofilm model with substrate inhibition is proposed for the activated sludge growing discs of rotating biological contactor (RBC); this model is different from the steady-state biofilm model based on the Monod assumption. Both deep and shallow types of biofilms are examined and discussed. The biofilm models based on both Monod and substrate inhibition (Haldane) assumptions are compared. In addition, the relationships between substrate utilization rate, biofilm thickness, and liquid phase substrate concentration are discussed. The influence order of the factors that affect the biofilm thickness is studied and discussed by combining the Taguchi method and grey relational analysis. In this work, a Taguchi orthogonal table is used to construct the series that is needed for grey relational analysis to determine the influence priority of the four parameters S _B , kX _f , K _s, and K _i .     

Biofilm model calibration and microbial diversity study using Monte Carlo simulations

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single‐species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single‐species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single‐species biofilm model was then extended to a multi‐species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/K_S). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution. Biotechnol. Bioeng. 2013; 110: 1323–1332. © 2012 Wiley Periodicals, Inc.     

Carbon Conversion Efficiency and Limits of Productive Bacterial Degradation of Methyl tert-Butyl Ether and Related Compounds

The utilization of the fuel oxygenate methyl tert-butyl ether (MTBE) and related compounds by microorganisms was investigated in a mainly theoretical study based on the Y_ATP concept. Experiments were conducted to derive realistic maintenance coefficients and K_s values needed to calculate substrate fluxes available for biomass production. Aerobic substrate conversion and biomass synthesis were calculated for different putative pathways. The results suggest that MTBE is an effective heterotrophic substrate that can sustain growth yields of up to 0.87 g g⁻¹, which contradicts previous calculation results (N. Fortin et al., Environ. Microbiol. 3:407-416, 2001). Sufficient energy equivalents were generated in several of the potential assimilatory routes to incorporate carbon into biomass without the necessity to dissimilate additional substrate, efficient energy transduction provided. However, when a growth-related kinetic model was included, the limits of productive degradation became obvious. Depending on the maintenance coefficient m_s and its associated biomass decay term b, growth-associated carbon conversion became strongly dependent on substrate fluxes. Due to slow degradation kinetics, the calculations predicted relatively high threshold concentrations, S_min, below which growth would not further be supported. S_min strongly depended on the maximum growth rate μ_max, and b and was directly correlated with the half maximum rate-associated substrate concentration K_s, meaning that any effect impacting this parameter would also change S_min. The primary metabolic step, catalyzing the cleavage of the ether bond in MTBE, is likely to control the substrate flux in various strains. In addition, deficits in oxygen as an external factor and in reduction equivalents as a cellular variable in this reaction should further increase K_s and S_min for MTBE.     

A new method to solve a non-steady-state multispecies biofilm model

A transient multispecies model for quantifying microbial space competition in biofilm is derived from existing models, introducing a new approach to biomass detachment modelling. This model includes inert biomass, substrate diffusion and utilization rate within the biofilm and diffusional layers. It predicts the evolution of biofilm thickness, bulk substrate concentration, species distribution and substrate concentration within the biofilm. A zero-dimensional transient model is described. Its steady-state solution is used to set up initial conditions of the one-dimensional model and case computation towards steady-state solution. Some numerical tools have been developed, enabling fast computation on microcomputers. Simulations show the validity of a zero-dimensional model and perturbated systems are also simulated. Simulations with experimental data give acceptable results.     

Membrane enzyme reactor with simultaneous separation using electrophoresis

A membrane enzyme reactor with simultaneous separation was investigated. Enzymes, urease and aspartase, were immobilized by a porous polytetrafluoroethylene membrane. Electrical field was applied in the medium while the reaction was carried out. Products with electrical charge could be separated through the membrane from the reaction medium as they were formed. Reaction behavior was analyzed by a simple model considering both pore-migration and reaction in the skelton of the membrane. According to the analysis the inherent reaction rate of the immobilized enzymes decreases significantly. This is probably caused by the structural variation of enzymes. For the case of urease, the change of pH inside the membrane may also cause the decrease of the reaction rate. The model analysis showed that the enzyme content in the membrane and the residence time of the substrate in the membrane governed overall extent of reaction.List of Symbols e g (dm³)^–1 enzyme concentration in the membrane - L cm membrane thickness - K _m mM Michaelis constant - Rate mmol · min^–1 · g^–1 rate of product formation per unit weight of enzyme - S mM substrate concentration - S _in mM inlet substrate concentration - S _out mM outlet substrate concentration - u cm · min^–1 migration rate - V V voltage between the electrodes - V _m mmol · min^–1 · g^–1 maximum reaction rate - X conversion - z cm distance from the surface inside the membrane - void fraction of the porous membrane - tortuosity of the membrane - min space time     

Solid support membrane‐aerated catalytic biofilm reactor for the continuous synthesis of (S)‐styrene oxide at gram scale

Catalytic biofilms minimize reactant toxicity and maximize biocatalyst stability in selective transformations of chemicals to value‐added products in continuous processes. The scaling up of such catalytic biofilm processes is challenging, due to fluidic and biological parameters affording a special reactor design affecting process performance. A solid support membrane‐aerated biofilm reactor was optimized and scaled‐up to yield gram amounts of (S)‐styrene oxide, a toxic and instable high value chemical synthon. A sintered stainless steel membrane unit was identified as an optimal choice as biofilm substratum and for high oxygen mass transfer. A stable expanded polytetrafluoroethylene (ePTFE) membrane was best suited for in situ substrate delivery and product extraction. For the verification of scalability, catalytic biofilms of Pseudomonas sp. strain VLB120ΔC produced (S)‐styrene oxide to an average concentration of 390 mM in the organic phase per day (equivalent to 24.4 g L_aq^–1 day^–1). This productivity was gained by efficiently using the catalyst with an excellent product yield on biomass of 13.6 g_product g_biomass^–1. This product yield on biomass is in the order of magnitude reported for other continuous systems based on artificially immobilized biocatalysts and is fulfilling the minimum requirements for industrial biocatalytic processes. Overall, 46 g of (S)‐styrene oxide were produced and isolated (purity: 99%; enantiomeric excess [ee]: >99.8%. yield: 30%). The productivity is in a similar range as in comparable small‐scale biofilm reactors highlighting the large potential of this methodology for continuous bioprocessing of bulk chemicals and biofuels.     

Modeling and analysis of layered stationary anaerobic granular biofilms

A model that portrays substrate profiles in a steady-state multispecies granular biofilm is developed and coupled with a biofilm detachment model. The model accounts for glucose, propionate, hydrogen, and acetate transformations performed by three bacterial trophic groups: acidogens, syntrophic bacterial consortia, and methanogens. This model adequately describes the phenomenon of propionate degradation under thermodynamically unfavorable bulk hydrogen concentrations. Also suggested is the superiority of the layered biofilm structure over homogeneous distribution of the trophic groups for anaerobic degradation of organic compounds. Furthermore, model analysis suggests that with increasing bulk glucose concentration biofilm thickness reaches a maximum that is then followed by biofilm disintegration. These results may have an important impact on the design and control of upflow anaerobic sludge bed reactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 122-130, 1997.     

Performance improvement of raw water pretreatment process with pre-inoculation biofilm: feasibility and limiting factors

The initial formation of biofilm and the removal performance of pollutants in biological pretreatment process for polluted raw water were limited due to the oligotrophic niche in raw water. In this study, the feasibility of using pre-inoculation biofilm formed under nutrients enhanced condition for polluted raw water treatment was analyzed in nine batch reactors. Results showed that the pollutants removal performance of biofilm was improved under nutrients enhanced conditions. Ammonia oxidation rate (AOR) was exponentially increased with the increasing in NH₄ ⁺-N levels, and organic matter removal rate (ORR) was positively related to the initial total organic carbon (TOC) concentration. The biofilm formation and microbial diversity were further improved via adding more substrates, seeding river sediment and feeding effluent from a mature biofilm reactor. However, the biofilm formed under higher substrate conditions had higher half-saturation constant values (K _S) to both NH₄ ⁺-N and TOC, which decreased AOR and ORR values when it was used to treat polluted raw water. The reduction percentage of AOR and ORR showed logarithmic growth modes with the increase in K _S values. Fortunately, improvement of nutrients flux via accelerating influent replacement could enhance the start-up performance effectively and decrease the operation risk introduced by the changes in substrate affinity.     

Fluidized bed biomethanation of acetic acid

Summary The kinetics of acetate biomethanation was studied in a high recycle ratio biological fluidized bed reactor behaving in practice as a completely mixed reactor. The active biofilm consisted of bacteria from a methane fermenter that after spontaneous immobilization on the bed particles (sand) were adapted to acetate as the only carbon source. The effects of temperature (13°, 20°, 25° and 35°C), substrate concentration (500, 1000 and 1500 mg chemical oxygen demand (COD) l^-1) and hydraulic retention time (1 to 8 h) on substrate consumption were studied. Maximum substrate consumption (as % COD reduction) amounted from 25% (13°C, 1500 mg COD l^-1) to 93% (35°C, 500 mg COD l^-1). At 35°C the concentration of attached biomass presented a weakly increase with reactor substrate concentration (from 3.10 g VS l^-1 to 4.54 g VS l^-1 for 32 and 1150 mg COD l^-1 respectively). On the other hand when reducing , a sharp incrase in biomass loss coefficient was observed showing that excess biofilm growth was continuously removed by shearing forces. Thus in the assayed conditions the attached biomass concentration was basically determined by the bed superficial velocity. Result show that diffusional resistances are negligible. Data are fairly well correlated by a variable order kinetic model. The apparent reaction order is a function of temperature and increases from 0.27 to 0.7 when temperature decreases from 35° C to 13°C.Nomenclature b Total biomass loss coefficient (T^-1) - J Flux of substrate removal into the biofilm surface (ML^-2 T^-1) - J _d Flux of substrate removed into the biofilm surface in deep conditions (ML^-2 T^-1) - k Maximum specific rate of substrate utilization (T^-1) - K Variable order kinetic constant (T^-1 M^n-1 L^3n-3) - K _s9 Hall saturation constant (ML^-3) - n Reaction order - q Feed flow rate (L³ T^-1) - S Substrate concentration (ML^-3) - Se Effluent substrate concentration (ML^-3) - So Influent substrate concentration (ML^-3) - Se_min Minimum substrate concentration able to sustain a steady-state biofilm (ML^-3) - T Temperature - t Time(T) - V Bed volume (L³) - VS Volatile solids (M) - VSS Volatile suspended solids - X Attached biomass concentration (ML^-3) - X _c Effluent volatile suspended solids (ML^-3) - Y Yield coefficient - Hydraulic retention time (T) This work forms part of a Doctoral Thesis of senior author     

Hydrocarbon fermentation: Kinetics of microbial cell growth

Modeling of microbial growth using nonmiscible substrate is studied when kinetics of substrate dissolution is rate limiting. When the substrate concentration is low, the growth rate is described by an analytical relation that can be identified as a Contois relationship. If the substrate concentration is greater than a critical value S_crit, the potentially useful hydrocarbon S* concentration is described by S* = S_crit/(1 + S_crit/S). A relationship was found between S_crit and the biomass concentration X. When X increased, S_crit decreased. The cell growth rate is related to a relation μ = μ_m[A(X/S_crit)(1 + S_crit/S) + 1]^?1. This model describes the evolution of the growth rate when exponential or linear growth occurs, which is related to physico-chemical properties and hydrodynamic fermentation conditions. Experimental data to support the model are presented.     

Simplified Modeling of Simultaneous Reaction Kinetics of Carbon Oxidation and Nitrification in Biofilm Processes

Batch experiments with varying initial substrate concentrations and biomass volumes were performed in a three‐phase fluidized bed biofilm reactor treating simulated domestic wastewater to study the simultaneous carbon oxidation and nitrification in the biofilm process. A simplified mass balance equation for the biofilm was proposed and five different kinetic rate equations were used to match the actual data. The kinetic parameters were obtained by nonlinear regression analysis on a set of two differential equations representing the simultaneous carbon oxidation and nitrification. The competitive inhibition model incorporating the effects of total organic carbon (TOC) concentrations on nitrification rates was the best‐suited model based on the average r². In this model, oxygen concentration and its affinity constants were not included. Instead, it was assumed that the rate of carbon oxidation is independent of the NH₄⁺‐N, while nitrification is affected by TOC. The number of parameters was successfully minimized without reducing its ability to accurately predict the bulk concentration time course, which would reduce computational complexity and possibly enhance the availability for an actual wastewater treatment process.     

A Model for the Biofiltration of Butyl Acetate and Xylene Mixtures by a Trickle‐Bed Air Biofilter

A mathematical model that incorporates the rates of the mass transfer process and the biofilm reaction is presented to predict the performance of a trickle‐bed air biofilter (TBAB) for treating butyl acetate and xylene mixtures. A thorough understanding of the factors that influence these rates is necessary before the practical application of a TBAB for treating many kinds of pure and mixed volatile organic compounds (VOC) in the air stream. The model presented consists of a set of mass balance equations for butyl acetate, xylene and oxygen in the bulk gas phase and within the biofilm. The butyl acetate and xylene concentration profiles of the gas phase predicted by the model were in good agreement with the measured data documented in a previous study. The most relevant parameters were evaluated in a sensitivity analysis to determine their respective effects on the model performance. Four parameters were identified to strongly influence the model performance, the surface area of the biofilm per volume unit of the packing material (A_S), the empty‐bed residence time (EBRT), the maximum specific growth rate of the microorganism (μ_m), and the microbial yield coefficient (Y). The practical application of the model to derive the performance equation is also presented and discussed. This equation makes it possible to simultaneously obtain a relatively high VOC removal efficiency and to minimize the capital cost.     

Influence of the ratio of the initial substrate concentration to biomass concentration on the performance of a sequencing batch reactor

Biomass behaviour and COD removal in a benchscale activated sludge reactor have been studied alternating anaerobic and aerobic conditions. Particular attention has been paid to the influence of the ratio of the initial substrate concentration (S ₀) to the initial biomass concentration (X ₀) on the reactor performance. Tests at very low ratios (S ₀/X ₀<2) demonstrate the existence of a threshold below which the reactor performance is seriously affected (S ₀/X ₀=0.5). Under conditions of total suppression of cell duplication, substrate maintenance requirements have also been calculated for the microbial consortium present in the activated sludges. The results obtained show that stressed biomass can survive conditions of substrate lack better than unstressed biomass.List of Symbols b h^–1 specific death rate - COD g/l chemical oxygen demand - DO g/l dissolved oxygen concentration - K _s g/l Monod saturation constant - MLSS g/l mixed liquor suspended solid concentration - P g/l phosphorus concentration - S g/l substrate concentration - S ₀ g/l initial substrate concentration - SS g/l suspended solid concentration - t h time - X g/l biomass concentration - X ₀ g/l initial biomass concentration - Y _SX g/g yield of growth on substrate - _max h^–1 maximum specific growth rate     

Organic Matter Degradation Kinetics in an Anaerobic Thermophilic Fluidised Bed Bioreactor

This paper describes the thermophilic anaerobic biodegradation of wine distillery wastewater (vinasses) in a laboratory fluidised bed reactor (AFB) with a porous support medium. The experimental protocol was defined to examine the effect of increasing organic loading rate on the efficiency of AFB and to report on its steady-state performance. Moreover, in order to evaluate treatment efficiency and to investigate fermentation kinetics in an AFB reactor, experimental data were used to estimate the ‘active biomass’ concentration using an autocatalytic kinetic model proposed in this paper, since viable biomass in AFB reactors is very difficult to measure experimentally. The AFB reactor was subjected to a program of steady-state operation over a range of hydraulic retention time (HRTs) of 2.5–0.37 days and organic loading rate (OLRs) up to 5.88 kgCOD/m³/day in order to evaluate its treatment capacity. The AFB reactor was initially operated with organic loading rate of 5.88 kgCOD/m³/day and HRT of 2.5 days. The chemical oxygen demand (COD) removal efficiency was found to be 96.5% in the reactor while the methane content of biogas produced in the digester reached 1.08 m³/m³digester/day. Over 94 days operating period, an OLR of 32 kgCOD/m³/day at a food-to-micro-organisms (F:M) ratio of 0.55 kgCOD/kgVS_att/day was achieved with 81.5% COD removal efficiency in the experimental AFB reactor. At this moment, the methane content of biogas produced in the digester reached 9.0 m³/m³digester/day. The proposed kinetic model is able to estimate kinetic constants of the biodegradation process: non-biodegradable substrate (S_nb) and active adhered biomass concentration (X_a). The parameters of the model were obtained by the curve-fitting method to the proposed kinetic model using the COD as substrate of the anaerobic process and assuming a maximum specific μ_max: 0.72 per day. The comparison of the measured concentration of volatile attached solids (VS_att) with the estimated ‘active’ biomass concentration indicated that extremely high ‘active biomass’ concentrations can be maintained in the system because biofilm thickness is limited by the liquid flow rate applied. This is due to the fact that the anaerobic fluidised bed system retains the growth support medium in suspension by drag forces exerted by upflowing wastewater, and the distribution of biomass holdup (in the form of a biofilm) is thus relatively uniform.     

Distributivity and processivity in multisite phosphorylation can be distinguished through steady-state invariants

Multisite protein phosphorylation and dephosphorylation are key cellular regulatory mechanisms but their system properties have been difficult to study in vivo and in vitro. Here we show by mathematical analysis that steady-state invariants enable the mechanism of the kinase or the phosphatase to be determined from steady-state measurements. Invariants exist when both enzymes act distributively (i.e., nonprocessively), making at most one modification in each molecular encounter. For instance, in the sequential case, in any experiment involving the same ingredients, the quantity [S_i−1][S_i+1]/[S_i]² always has the same value, where [S_i] denotes the steady-state concentration of the i-th phospho-form. For a two-site substrate, if either enzyme exhibits processivity, so that more than one modification can be made in each molecular encounter, the degree of processivity can be estimated from changes in this invariant. We discuss the experimental and theoretical challenges in extending these results.     

Simulation of a multicolumn recirculated packed bed reactor (MRPBR) for penicillin acylase

Enzyme reactors for the industrial hydrolysis of penicillin are analyzed in terms of biocatalyst stability to pH. A multicolumn system with packed beds placed in parallel and operating under recirculating conditions is proposed as an adequate reactor for this process. The system is studied both experimentally and with the aid of a simulation program.List of Symbols A transversal area (cm²) - C _A ammonia concentration in the reaction mixture (M) - C ₁ concentration of KH₂PO₄ in buffer (M) - C ₂ concentration of K₂HPO₄ in buffer (M) - d _p biocatalyst diameter (cm) - E enzyme or biocatalyst concentration (gcat l^–1) - K _APA APA non competitive inhibition constant (M) - K _IS excess substrate inhibition constant (M) - Km constant Michaelis-Menten (M) - K _PAA PAA competitive inhibition constant (M) - Q recirculation flow rate (cm³ min^–1) - Q _T recirculation flow rate per column (cm³ min^–1) - Re Reynolds number - S _E substrate concentration entering the neutralization tank (M) - S ₀ initial substrate concentration (M) - S _T substrate concentration in neutralization tank (M) - t time (min) - v _i initial reactor rate (mol min^–1 gcat^–1) - V _s superficial velocity (cm seg^–1) - V _T volume of neutralization tank (cm³) - X _E substrate conversion entering tank - X _T substrate conversion in neutralization tank - X conversion - Z reactor length (cm) - z axial position in reactor (cm) - z ^* non-dimensional axial position in reactor - biocatalyst's density (gcat cm^–3) - p pressure drop in the packed-bed reactor     

A cybernetic model to predict the effect of freely available nitrogen substrate on rifamycin B production in complex media

It is well-known that secondary metabolite production is repressed by excess nitrogen substrate available in the fermentation media. Although the nitrogen catabolite repression has been known, quantitative process models have not been reported to represent this phenomenon in complex medium. In this paper, we present a cybernetic model for rifamycin B production via Amycolatopsis mediterranei S699 in complex medium, which is typically used in industry. Nitrogen substrate is assumed to be present in two forms in the medium; available nitrogen (S _ANS) such as free amino acids and unavailable nitrogen (S _UNS) such as peptides and proteins. The model assumes that an inducible enzyme catalyzes the conversion of S _UNS to S _ANS. Although S _ANS is required for growth and product formation, high concentrations were found to inhibit rifamycin production. To experimentally validate the model, five different organic nitrogen sources were used that differ in the ratio of S _ANS/S _UNS. The model successfully predicts higher rifamycin B productivity for nitrogen sources that contain lower initial S _ANS. The higher productivity is attributed to the sustained availability of S _ANS at low concentration via conversion of S _UNS to S _ANS, thereby minimizing the effects of nitrogen catabolite repression on rifamycin production. The model can have applications in model-based optimization of substrate feeding recipe and in monitoring and control of fed batch processes.     

Production of elemental sulfur by green and purple sulfur bacteria

The utilization of sulfide by phototrophic sulfur bacteria temporarily results in the accumulation of elemental sulfur. In the green sulfur bacteria (Chlorobiaceae), the sulfur is deposited outside the cells, whereas in the purple sulfur bacteria (Chromatiaceae) sulfur is found intracellularly. Consequently, in the latter case, sulfur is unattainable for other individuals. Attempts were made to analyze the impact of the formation of extracellular elemental sulfur compared to the deposition of intracellular sulfur.According to the theory of the continuous cultivation of microorganisms, the steady-state concentration of the limiting substrate is unaffected by the reservoir concentration (S _R).It was observed in sulfide-limited continuous cultures ofChlorobium limicola f.thiosulfatophilum that higherS _R values not only resulted in higher steady-state population densities, but also in increased steady-state concentrations of elemental sulfur. Similar phenomena were observed in sulfide-limited cultures ofChromatium vinosum.It was concluded that the elemental sulfur produced byChlorobium, althouth being deposited extracellularly, is not easily available for other individuals, and apparently remains (in part) attached to the cells. The ecological significance of the data is discussed.Non-standard abbreviations RP reducing power - BChl bacteriochlorophyll - N_cell cell material - specific growth rate - {ie52-1} maximal specific growth rate - D dilution rate - K _s saturation constant - s concentration of limiting substrate - S _R same ass but in reservoir bottle - Y yield factor - iS^o intracellular elemental sulfur - eS^o extracellular elemental sulfur - PHB poly-beta-hydroxybutyric acid     

A model for treating toluene and acetone mixtures by a trickle-bed air biofilter

A mathematical model that incorporates mass transfer process and biofilm reactions is presented to predict the performance of a trickle-bed air biofilter (TBAB) for treating toluene (T) and acetone (ACE) mixtures. The model consists of a set of mass balance equations for T, ACE and oxygen in the bulk gas phase and within the biofilm. The gas phase T and ACE concentrations predicted by the model were in good agreement with the measured data available in a previous study. The important parameters were evaluated in the sensitivity analysis to determine their respective effects on the model performance. Four parameters were identified as strongly influencing the model performance, the surface area of the biofilm per unit volume of packing material (A _S), the empty-bed residence time (EBRT), the maximum specific growth rate of microorganism ( _m), and the microbial yield coefficient (Y). A practical application of the model to derive the performance equation of TBAB is also given.