Kinetic and stoichiometric characterization of a fixed biofilm reactor by pulse respirometry

An in situ respirometric technique was applied to a sequential biofilm batch reactor treating a synthetic wastewater containing acetate. In this reactor, inoculated with mixed liquor from a wastewater plant, unglazed ceramic tiles were used as support media while maintaining complete mixing regime. A total of 8 kinetic and stoichiometric parameters were determined by in situ pulse respirometry; namely substrate oxidation yield, biomass growth yield, storage yield, storage growth yield, substrate affinity constant, storage affinity constant, storage kinetic constant and maximum oxygen uptake rate. Additionally, biofilm growth was determined from support media sampling showing that the colonization process occurred during the first 40 days, reaching an apparent steady-state afterward. Similarly, most of the stoichiometric and kinetic parameters were changing over time but reached steady values after day 40. During the experiment, the respirometric method allowed to quantify the amount of substrate directed to storage, which was significant, especially at substrate concentration superior to 30 mg COD L⁻¹. The Activated Sludge Model 3 (ASM3), which is a model that takes into account substrate storage mechanisms, fitted well experimental data and allowed confirming that feast and famine cycles in SBR favor storage. These results also show that in situ pulse respirometry can be used for fixed-bed reactors characterization.     

Comparison of static and dynamic respirometry for the determination of stoichiometric and kinetic parameters of a nitrifying process

Respirometry consists in the measurement of the biological oxygen consumption rate under well-defined conditions and has been used for the characterization of countless biological processes. In the field of biotechnology and applied microbiology, several respirometry methods are commonly used for the determination of process parameters. Dynamic and static respirometry, which are based on oxygen measurements with or without continuous aeration, respectively, are the methods most commonly used. Additionally to several respirometry methods, different methods have also been developed to retrieve process parameters from respirometric data. Among them, methods based on model fitting and methods based on the injection of substrate pulse at increasing concentration are commonly used. An important question is then; what respirometry and data interpretation methods should be preferably used? So far, and despite a growing interest for respirometry, relatively little attention has been paid on the comparison between the different methods available. In this work, both static and dynamic respirometry methods and both interpretation methods; model fitting and pulses of increasing concentration, were compared to characterize an autotrophic nitrification process. A total of 60 respirometry experiments were done and exhaustively analysed, including sensitivity and error analyses. According to the results obtained, the substrate affinity constant (K _S ) was better determined by static respirometry with pulses of increasing concentration and the maximum oxygen uptake rate (OUR _ex.max ) was better determined by dynamic respirometry coupled to fitting procedure. The best method for combined K _S and OUR _ex.max determination was static respirometry with pulses of increasing concentration.     

A critical comparison of extant batch respirometric and substrate depletion assays for estimation of nitrification biokinetics

Estimation of nitrification biokinetics has been conducted using different batch techniques via measurement of nitrogen species or surrogates such as oxygen (respirometry). However, there are no reports that specifically compare kinetic parameters estimated from respirometry with those from direct nitrogen species measurements. In this study, we evaluated the ability of parameter estimates from isolated and optimally designed complete extant respirometric assays to describe concurrently obtained ammonia and nitrite depletion profiles. Additionally, we mapped the different parameter sets to steady-state bioreactor performance. Using multivariate analysis of variance, we found that estimates from respirometric and substrate depletion assays were predominantly statistically different at the 95% confidence level. The sensitivity of predicted stead-state nitrifying reactor performance to differences in parameter estimates was highest close to the limiting solids retention time (SRT). However, at characteristic nitrifying SRTs the predicted reactor performance using parameter estimates from respirometry and substrate depletion assays were in very close correspondence. Therefore, parameters estimated from extant respirometric assays can be used to adequately predict nitrifying reactor performance.     

Microrespirometric characterization of activated sludge inhibition by copper and zinc

We have developed a novel microrespirometric method to characterize the inhibitory effects due to heavy metals on activated sludge treatment. This method was based on pulse dynamic respirometry and involved the injection of several pulses of substrate and inhibitors, of increasing concentration. Furthermore, we evaluated the inhibitory effects of heavy metals (copper and zinc), substrate and biomass concentrations, and pH on activated sludge activity. While higher biomass concentrations counteracted the inhibitory effects of both copper and zinc, higher substrate concentrations predominantly augmented the inhibitory effect of copper but no significant change in inhibition by zinc was observed. pH had a clear but relatively small effect on inhibition, partially explained by thermodynamic speciation. We determined the key kinetic parameters; namely, the half saturation constant (K _S ) and the maximum oxygen uptake rate (OUR _max ). The results showed that higher heavy metal concentrations substantially decreased K _S and OUR _max suggesting that the inhibition was uncompetitive.     

Kinetic and stoichiometric parameters estimation in a nitrifying bubble column through "in-situ" pulse respirometry

This article proposes a simple "in-situ" pulse respirometric method for the estimation of four important kinetic and stoichiometric parameters. The method is validated in a suspended biomass nitrifying reactor for the determination of (i) maximum oxygen uptake rate (OUR(ex)max), (ii) oxidation yield (f(E)), (iii) biomass growth yield (f(S)), and (iv) affinity constant (K(S)). OUR(ex)max and f(E) were directly obtained from respirograms. In the presented case study, a minimum substrate pulse of 10 mgNH(4) (+)-N L(-1) was necessary to determine OUR(ex)max which was 61.15 +/- 4.09 mgO(2) L(-1) h(-1) (5 repetitions). A linear correlation (r(2) = 0.93) obtained between OUR(ex)max and the biomass concentration in the reactor suggests that biomass concentration can be estimated from respirometric experiments. The substrate oxidation yield, f(E), was determined along 60 days of continuous operation with an average error of 5.6%. The biomass growth yield was indirectly estimated from the substrate oxidation yield f(E). The average obtained value (0.10 +/- 0.04 mgCOD mg(-1)COD) was in accordance with the f(S) estimation by the traditional COD mass balance method under steady-state conditions (0.09 +/- 0.01). The affinity constant K(S) was indirectly estimated after fitting the ascending part of the respirogram to a theoretical model. An average value of 0.48 +/- 0.08 mgNH(4) (+)-N L(-1) was obtained, which is in the range of affinity constants reported in the literature for the nitrification process (0.16-2 mgNH(4) (+)-N L(-1)).     

Biodegradation kinetics of stored wastewater substrates by a mixed microbial culture

The anaerobic accumulation of several organic pollutants from industrial wastewaters, as storage substrates, and their subsequent aerobic biodegradation using a wastewater treatment mixed microbial culture for biological nutrient removal has been studied. The amount and the kinetics of substrate accumulation in the anaerobic stage depended on the characteristics of the wastewater fed to the anaerobic stage. Depending on the substrate used, levels of between 27 and 86% of storage polymers were accumulated with respect to the level obtained on feeding with acetate. The biodegradation kinetics were studied by modelling respirometry results. During the aerobic stage, oxygen-consumption data obtained in the respirometric tests were fitted to a model using a non-linear fitting estimation method. The simulation data obtained correlated well with the experimental oxygen-consumption data. The estimated kinetic parameters obtained indicate that each storage polymer was degraded at a different rate. However, the values obtained for the storage polymer half-saturation coefficient, K_S: 16 mg COD l⁻¹, and for the coefficient for endogenous respiration, b: 0.008 h⁻¹, were similar in all the experiments. The results indicate that each substrate produces the synthesis of a specific storage polymer that is degraded at a different rate.     

Estimation of Nitrifying Bacterial Activities by Measuring Oxygen Uptake in the Presence of the Metabolic Inhibitors Allylthiourea and Azide

The effects of two metabolic inhibitors on an enriched nitrifying biomass during incubation for short periods of time were investigated by determining respirometric measurements. Allylthiourea (86 μM) and azide (24 μM) were shown to be strong, selective inhibitors of ammonia and nitrite oxidation, respectively. Consequently, a differential respirometry method for estimating nitrifying and heterotrophic bacterial activities within a mixed biomass is proposed.     

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 μg of mitochondria indicate that 1.0 μg is sufficient to measure respiration and that 5.0 μg provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.     

Characterization of tannery wastewater and biomass in a membrane bioreactor using respirometric analysis

Respirometric techniques and an activated sludge model (ASM) were applied for the characterization of tannery wastewater and biomass in a pilot plant membrane bioreactor (MBR) operating at high sludge age. The traditional respirometric tests and the IWA-ASM1 were modified to take into account the specific operating conditions, the solid-liquid separation technology and the wastewater complexity. As a result the wastewater biodegradable COD was fractionated into four components: readily biodegradable, rapidly hydrolysable, slowly hydrolysable and inorganic (due to the presence of reduced sulphur compounds). The kinetic and stoichiometric parameters of the biomass (heterotrophic and nitrifying) were estimated through the integration of model simulations and respirometric tests results. In particular the ammonium and nitrite-oxidizing biomasses were separately characterized: the growth kinetics of ammonium and nitrite-oxidizing bacteria resulted noticeably lower than the traditional reference values (mu(max,AOB)=0.25d(-1)e mu(max,NOB)=0.23d(-1) at 20 degrees C, respectively). The ASM was finally used to confirm that the results of the wastewater and biomass characterization allow to properly simulate the mixed liquor suspended solids in the MBR pilot plant and the COD concentration in the effluent.     

Respirometric response and microbial succession of nitrifying sludge to <Emphasis Type="Italic">m</Emphasis>-cresol pulses in a sequencing batch reactor

A nitrifying consortium was kinetically, stoichiometrically and molecularly characterized via the in situ pulse respirometric method and pyrosequencing analysis before and after the addition of m-cresol (25 mg C L^?1) in a sequencing batch reactor (SBR). Five important kinetic and stoichiometric parameters were determined: the maximum oxygen uptake rate, the maximum nitrification rate, the oxidation yield, the biomass growth yield, and the substrate affinity constant. An inhibitory effect was observed in the nitrification process with a recovery of this by up to eight SBR cycles after m-cresol was added to the system. However, full recovery of the nitrification process was not observed, as the maximum oxygen uptake rate was 25% lower than that of the previous operation without m-cresol addition. Furthermore, the pyrosequencing analyses of the nitrifying consortium after the addition of only two pulses of 25 mg C L^?1 m-cresol showed an important microbial community change represented by a decrease in the nitrifying populations and an increase in the populations degrading phenolic compounds.     

Comparison of experimental methods for determination of toxicity and biodegradability of xenobiotic compounds

Different methods for determining the toxicity and biodegradability of hazardous compounds evaluating their susceptibility to biological treatment were studied. Several compounds including chlorophenols and herbicides have been evaluated. Toxicity was analyzed in terms of EC₅₀ and by a simple respirometric procedure based on the OECD Method 209 and by the Microtox^® bioassay. The values of EC₅₀ obtained from respirometry were in all the cases higher than those from the Microtox^® test. The respirometric inhibition values of chlorophenols were related well with the number of chlorine atoms and their position in the aromatic ring. In general, herbicides showed lower inhibition, being alachlor the less toxic from this criterion. For determination of biodegradability an easier and faster alternative to the OECD Method 301, with a higher biomass to substrate ratio is proposed. When this test was negative, the Zahn-Wellens one was performed in order to evaluate the inherent biodegradability. In the fast test of biodegradability, 4-chlorocatechol and 4-chlorophenol showed a complete biodegradation by an unacclimated sludge upon 48 h. These results together with their low respirometric inhibition, allow concluding that these compounds could be conveniently removed in a WWTP. Alachlor, 2,4-dichlorophenol, 2,4,6-trichlorophenol and MCPA showed a partial biodegradation upon 28 days by the Zahn-Wellens inherent biodegradability test.     

Adapting High-Resolution Respirometry to Glucose-Limited Steady State Mycelium of the Filamentous Fungus Penicillium ochrochloron: Method Development and Standardisation

Fungal electron transport systems (ETS) are branched, involving alternative NADH dehydrogenases and an alternative terminal oxidase. These alternative respiratory enzymes were reported to play a role in pathogenesis, production of antibiotics and excretion of organic acids. The activity of these alternative respiratory enzymes strongly depends on environmental conditions. Functional analysis of fungal ETS under highly standardised conditions for cultivation, sample processing and respirometric assay are still lacking. We developed a highly standardised protocol to explore in vivo the ETS—and in particular the alternative oxidase—in Penicillium ochrochloron. This included cultivation in glucose-limited chemostat (to achieve a defined and reproducible physiological state), direct transfer without any manipulation of a broth sample to the respirometer (to maintain the physiological state in the respirometer as close as possible to that in the chemostat), and high-resolution respirometry (small sample volume and high measuring accuracy). This protocol was aimed at avoiding any changes in the physiological phenotype due to the high phenotypic plasticity of filamentous fungi. A stable oxygen consumption (< 5% change in 20 minutes) was only possible with glucose limited chemostat mycelium and a direct transfer of a broth sample into the respirometer. Steady state respiration was 29% below its maximum respiratory capacity. Additionally to a rotenone-sensitive complex I and most probably a functioning complex III, the ETS of P. ochrochloron also contained a cyanide-sensitive terminal oxidase (complex IV). Activity of alternative oxidase was present constitutively. The degree of inhibition strongly depended on the sequence of inhibitor addition. This suggested, as postulated for plants, that the alternative terminal oxidase was in dynamic equilibrium with complex IV—independent of the rate of electron flux. This means that the onset of activity does not depend on a complete saturation or inhibition of the cytochrome pathway.     

Multiple analysis reprogrammable titration analyser for the kinetic characterization of nitrifying and autotrophic denitrifying biomass

The system Multiple Analysis Reprogrammable TItratioN Analyser (MARTINA) based on titrimetric techniques has been used to kinetically characterize different types of sludges: nitrifying, enriched ammonia oxidizing and autotrophic denitrifying biomass. The titration system employed, combines the addition of NaOH solution and H₂O₂ solution in the mixed liquor to keep the pre-established value of pH and the dissolved oxygen concentration, respectively. Results obtained from repeated experiments performed with nitrifying sludge from municipal and industrial origin present slight differences (coefficient of variation lower than 30%) indicating that the method is highly reproducible. Besides, the kinetic parameters of the enriched ammonia oxidizing sludge obtained using the MARTINA system are comparable to those obtained using the respirometry indicating the reliability of this methodology. Changes in the procedure may be easily implemented in order to estimate half saturation constants with high values. On the other hand, experiments in anoxic conditions applied to the estimation of the kinetic parameters of the autotrophic denitrifying biomass have been successfully performed, even if this process involves a reaction characterized by slight pH changes. The titration system MARTINA is a reproducible, reliable, versatile and precise alternative to the traditional respirometric and substrate monitoring tests for the characterization of kinetics for a wide range of sludges in aerobic or anoxic conditions.     

The process utility of thermotolerant methylotrophic bacteria: II. An evaluation of transient responses

In Part II, the process utility of a thermotolerant methylotrophic bacterium is evaluated with respect to its dynamic response when various substrate or nutrient pulses are imposed on it while growing under steady-state conditions in a chemostat. The pulses investigated were methanol pulses on methanol-, methanol/formaldehyde-, and dual methanol/ammonia-limited cultures and an ammonia pulse on a severely nitrogen (ammonia)-limited culture. The results obtained, although exemplifying the complex biochemistry of such bacteria, clearly demonstrate the bacteriums flexibility and versatility in handling process transients. Its lack of fastidiousness in unsteady state continuous culture make it a most promising candidate for inclusion in process cultures for elevated temperature industrial wastewater treatment.     

Self-Cycling Fermentation Applied to Acinetobacter calcoaceticus RAG-1

The self-cycling fermentation (SCF) technique was applied to a culture of Acinetobacter calcoaceticus RAG-1. This method was shown to result in synchronization of the cells, achieving a 77% improvement in cell synchrony over that of the batch case. Cellular occurrences, averaged out by asynchronous batch cultures, were magnified by the temporal alignment of metabolic events brought about by the synchronization associated with SCFs. The cell population doubled only once per cycle, thus establishing an equality between cycle time and doubling time. Parameters of interest were biomass concentration, total bioemulsifier (emulsan) production, cycle time, and residual carbon concentration. Cycle-to-cycle variation of these parameters was, in most cases, insignificant. Repeatability of doubling time estimates (based on 95% confidence intervals) was roughly 7 to 10 times better between cycles in an SCF than between batch replicates. The carbon substrate was completely utilized in all cases in which it was measured, giving this technique an advantage over chemostat-type fermentations. The dissolved-oxygen profiles monitored throughout a cycle were found to be repeatable. A characteristic shape, which can be related to the growth of the organism, was associated with each carbon source. The specific emulsan productivity of SCFs was found to be approximately 50 times greater than that of the batch process and 2 to 9 times greater than that of the chemostat, depending on the dilution rate considered. With respect to specific emulsan production, a 25-fold improvement over that in an immobilized cell system recently introduced was obtained. Thus, SCFs are a viable alternative to established fermentation techniques.     

The continuously fed batch reactor for measuring microbial growth rates

The contiuously fed batch reactor (CFBR) is proposed as an alternative technique to the traditional chemostat and batch cultures, for measuring microbial growth rates. After reviewing the pitfalls which plague the conventional growth measurement techniques, the methodology for operating the CFBR to generate specific growth-rate-versus-substrate-concentration data is detailed. This information is extracted from the transient state of the CFBR where both the biomass and substrate concentration show extrema in time. It is suggested that the CFBR can be used for measuring microbial growth rates at low rates at low substrate concentrations where the chemostat method normally encounters difficulties.     

On-line monitoring of PHB production by mixed microbial cultures using respirometry,titrimetry and chemometric modelling

This work describes a method for on-line monitoring of biomass production, acetate consumption and intracellular polyhydroxybutyrate (PHB) storage by mixed microbial cultures (MMC). The method is based on reliable and easily available on-line measurements, namely pH, dissolved oxygen, dissolved carbon dioxide, on-line respirometry and on-line titrimetric analysis. Biomass production refers to active biomass growth and also to the synthesis of extracellular polymeric substances (EPS). The composition and kinetics of EPS synthesis has high variability depending on the culture enrichment protocol. Since the metabolism for EPS production is rather difficult to define, it was not possible to develop a reliable estimation model based on metabolic principles only. Instead, projection of latent structures (PLS) linear regression constrained by steady state carbon balance was employed. PHB concentration and biomass production rate were directly estimated by the PLS model, whereas acetate concentration was indirectly estimated through the carbon balance. The method was validated experimentally with data of four experiments carried out in a SBR. Accurate on-line estimations were obtained with regression coefficients (r²) of 0.986 and 0.980 for biomass concentration, 0.976 and 0.999 for PHB and 0.992 and 0.999 for acetate concentration in calibration and validation, respectively. These results confirm the ability of the proposed methodology for on-line monitoring of the state variables in PHB production process by MMC.     

Defined media optimization for growth of recombinant Escherichia coli X90

An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h(-1) for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture. (c) 1993 John Wiley & Sons, Inc.     

Noninvasive cardiac output estimation: a preliminary study

 The availability of a simple-to-use, automatic measurement system for noninvasive flow estimation is imperative, given the clinical demand for an acceptable noninvasive procedure rather than the standard invasive procedure of thermodilution. A method for calculating cardiac output from noninvasively derived pressure pulses has been developed, and the results of a preliminary evaluation study on post-cardiac surgery patients for whom invasive flow measures were readily available for comparison are provided in this report. The proposed method relies on fast Fourier transform (FFT) analysis of pulses measured externally at the carotid and femoral pressure points. A transfer function of the aorta is computed from digitally filtered pulse measurements, and a tapered model of the aorta is parametrically adapted using a simplex optimization algorithm so that its transfer function matches that derived experimentally. An aortic input impedance term is obtained from the optimized model and utilized along with the carotid pulse (analogous to input voltage) to compute aortic flow. In addition to its automation, attractive features of this method include the requirement for relatively few pulses for analysis as well as considerable resistance to noise artifact. For 59 data records collected from 54 post-cardiac surgery patients, the average flow measurements computed over several pulses compare well with the standard, invasive method of thermodilution. Preliminary results also indicate a strong potential for tracking changes in cardiac output over time, and invite further use of the method in monitoring hemodynamically unstable patients. Received: 18 February 1997 / Accepted in revised form: 12 May 1997