Specific Substrate Uptake Rate Research Articles

PAT method to gather bioprocess parameters in real-time using simple input variables and first principle relationships

The efficient quantification of bioprocess parameters is of high importance for strain characterization and process development. Here, we present a method, which allows the quantification of the specific growth rate, the specific substrate uptake rate and the growth stoichiometry, in a real-time context. The method is solely based on the combination of first principle relationships, using unstructured kinetic modeling, elemental- and mass balancing. Hence, very little prior knowledge is required. Off gas measurements of CO 2 and O 2 as well as base consumption were used as the only input variables to the method, which was executed in real-time along with the bioprocess. We demonstrate the capacity of the method by fed-batch cultivations of the methylotrophic yeast Pichia pastoris as well as by batch cultivations of P. pastoris and Saccharomyces cerevisiae on various carbon substrates. In fed-batch, with an accelerated and decelerated exponential feed profile, the method was able to efficiently quantify the maximum specific growth rate, as well as to investigate growth stoichiometry at low growth rates for maintenance effects. Furthermore, the method allows to correctly identify the timely adaptation of the specific substrate uptake rate and the specific growth rate as well as the oxidative capacity in batch cultures and to distinguish substrate specific kinetics. Further real-time outputs are concentrations of components such as biomass and substrate. The validity of the derived parameters is checked for consistency using redundancy and a statistical test. This method contributes to process understanding, because the method consistently quantifies bioprocess parameters, which can be used to analyze their correlation to critical quality attributes. Furthermore, the real-time availability of the parameters allows the development of process control strategies. This matches Quality by Design (QbD) and Process Analytical Technology (PAT) in its main objectives.

Evaluation of software sensors for on-line estimation of culture conditions in an Escherichia coli cultivation expressing a recombinant protein

Software sensors for monitoring and on-line estimation of critical bioprocess variables have mainly been used with standard bioreactor sensors, such as electrodes and gas analyzers, where algorithms in the software model have generated the desired state variables. In this article we propose that other on-line instruments, such as NIR probes and on-line HPLC, should be used to make more reliable and flexible software sensors. Five software sensor architectures were compared and evaluated: (1) biomass concentration from an on-line NIR probe, (2) biomass concentration from titrant addition, (3) specific growth rate from titrant addition, (4) specific growth rate from the NIR probe, and (5) specific substrate uptake rate and by-product rate from on-line HPLC and NIR probe signals. The software sensors were demonstrated on an Escherichia coli cultivation expressing a recombinant protein, green fluorescent protein (GFP), but the results could be extrapolated to other production organisms and product proteins. We conclude that well-maintained on-line instrumentation (hardware sensors) can increase the potential of software sensors. This would also strongly support the intentions with process analytical technology and quality-by-design concepts.

Evaluation of Influent Medium Composition and Concentration in a Batch Reactor for Effective Startup of UASB Reactors

This study demonstrated the use of batch bioreactor experiments as an evaluation tool to determine appropriate influent medium composition and concentration for effective startup of an upflow anaerobic sludge blanket (UASB) reactor. Using seed sludge from a domestic treatment plant, the weight ratio of glucose and volatile fatty acids (VFA) present in the influent synthetic medium was varied and the batch performance was assessed in terms of pH variation during the course of the experiment, chemical oxygen demand (COD) reduction, specific substrate uptake rates, methanogenic activity, and methane yield. The performance was also evaluated by increasing the concentration of influent COD. Medium composition with glucose to VFA weight ratio at or below 2:8 and COD concentration at 11g∕L was determined to be optimum for effective startup of the UASB reactors. Utilization of the optimized influent medium provided a pH variation from 6.5 to 7.8 over the length of the UASB reactor and resulted in granule formation, high methanogenic activity, and methane yield. The evaluation method provided a practical approach to determine the applicability of seed sludge from a particular source and the desired influent characteristics for reduced startup duration in UASB processes.

Citric acid production by a novel Aspergillus niger isolate: I. Mutagenesis and cost reduction studies

Ultraviolet-irradiation (UV), ethyl methane sulfonate (EMS) and acridine orange (AO) were used to induce citric acid overproduction mutations in Aspergillus niger UMIP 2564. Among 15, eight of the mutant derivatives, were improved with respect to citric acid production from sucrose in batch cultures. Maximum product yield (60.25%) was recorded by W5, a stable UV mutant, with approximately 3.2-fold increase when compared to the parental wild type strain. In terms of the kinetic parameters for batch fermentation processes, the mutation doubled the specific substrate uptake rate and achieved 4.5- and 7.5-fold improvements in citric acid productivity and specific productivity, respectively. For reduction of the fermentation medium cost, corn steep liquor and calcium phosphate pre-treated beet molasses were successfully used as substituents of nitrogen and carbon sources in the growth medium, respectively. These medium substitutions resulted in a W5 citric acid fermentation culture with a product yield of 74.56%.

Biodegradation of oxadiazon by a soil isolated Pseudomonas fluorescens strain CG5: Implementation in an herbicide removal reactor and modelling

An oxadiazon-degrading bacterial, Pseudomonas strain CG5, was isolated from an agricultural contaminated soil. This strain CG5 was able to grow on 10 mg of oxadiazon per l, yielding 5.18±0.2 mg of protein biomass mol −1. GC-MS analyses of the metabolites from oxadiazon catabolism revealed its dehalogenation and degradation to form non-toxic end-products, cells were then immobilized by adsorption on a ceramic support to be used as biocatalysts in herbicide removal biofilm-reactor processes. Seventy-two per cent of the oxadiazon was removed, and the maximum specific substrate uptake rate was 10.63±0.5 μg h –1 mg −1 prot. A new mathematical model was developed to interpret and predict the behaviour of the bacteria and pollutants in a biofilm-reactor system, to consider biofilm structural and morphological properties.

Kinetic models of an anaerobic bioreactor for restoring wastewater generated by industrial chickpea protein production

To assess the accuracy of kinetic models in predicting the behaviour of an anaerobic biodegradation process for cleaning up wastewater derived from the production of protein isolates from chickpea flour, the process was conducted in a laboratory-scale fluidised-bed reactor with saponite (magnesium silicate) as support for the mediating bacteria at 35 °C. The reactor operated satisfactorily at organic loading rates between 0.58 and 2.70 g total chemical oxygen demand (TCOD) L?1 d?1, hydraulic retention times between 14.9 and 3.5 d, and an average feed TCOD of 9.1 g L?1. The methane yield coefficient value, YG/S, of 0.33 L CH4 (at STP) g?1 TCODremoved, calculated on the basis of a substrate balance (TCOD) model, was virtually identical to that obtained from the experimental data. The cell maintenance coefficient, km, obtained by means of this balance was found to be 0.0057 g TCOD g?1 volatile suspended solids (VSS) d?1. The volumetric methane production rates correlated with the biodegradable TCOD concentration based on the Michaelis–Menten equation. In addition, the specific rate of substrate uptake, r (g soluble COD (SCOD) g?1 VSS d?1), also correlated with the concentration of biodegradable substrate, Sb (g SCOD L?1), according to the Michaelis–Menten equation. These proposed models predict the behaviour of the reactor accurately showing deviations <10% between experimental and theoretical values of methane production and substrate uptake rates, respectively.

Simulation of a membrane bioreactor for regeneration of degreasing systems

The dynamic behaviour of membrane bioreactors (MBRs) with negligible biomass growth due to the presence of toxic substances was modelled to simulate a pilot plant test in an industrial degreasing unit. Modelling was done by introducing a substrate biodegradation rate into a material balance equation of an MBR. This was followed by a discrimination procedure between the simulated and measured values, from which best-fit specific substrate uptake rates (qsp) were determined at different air supply rates. The results showed clear improvement of qsp with increased air supply, indicating a possible increase in the active cell population in the reactor due to reduced toxicity. The highest specific substrate uptake rate (qsp = 0.08 gsubstrate gbiomass−1 d−1) determined was assumed equal to the maintenance coefficient (qm) for the biodegradation of the substrate under the test conditions. The understanding of the dynamics provided a reference in subsequent regeneration system improvement. Copyright © 2006 Society of Chemical Industry

The dynamics of single-substrate continuous cultures: The role of ribosomes

When a chemostat is perturbed from its steady state, it displays complex dynamics. For instance, if the identity of the growth-limiting substrate is switched abruptly, the substrate concentration and cell density undergo a pronounced excursion from the steady state that can last several days. These dynamics occur because certain physiological variables respond slowly. In the literature, several physiological variables have been postulated as potential sources of the slow response. These include transport enzymes, biosynthetic enzymes, and ribosomes. We have been addressing this problem by systematically exploring the role of these variables. In previous work Shoemaker et al. (J. Theor. Biol., 222 (2003) 307–322), we studied the role of transport enzymes, and we showed that transients starting from low transport enzyme levels could be quantitatively captured by a model taking due account of transport enzyme synthesis. However, there is some experimental data indicating that slow responses occur even if the initial enzyme levels are high. Here, we analyse this data to show that in these cases, the sluggish response is most probably due to slow adjustment of the ribosome levels. To test this hypothesis, we extend our previous model by accounting for the evolution of both the transport enzyme and the ribosomes. Based on a kinetic analysis of the data in the literature, we assume that the specific protein synthesis rate is proportional to the ribosome level, and the specific ribosome synthesis rate is autocatalytic. Simulations of the model show remarkable agreement with experimentally observed steady states and the transients. Specifically, the model predictions are in good agreement with (1) the steady-state profiles of the cell density, substrate concentration, RNA, proteins, and transport enzymes, (2) the instantaneous specific substrate uptake, growth, and respiration rates in response to a continuous-to-batch shift, and (3) the transient profiles of the cell density, substrate concentration, and RNA in response to feed switches and dilution rate shifts. Time-scale analysis of the model reveals that every transient response is a combination of two fundamental (and simpler) dynamics, namely, substrate-sufficient batch dynamics and cell-sufficient fed-batch dynamics. We obtain further insight into the transient response by analysing the equations describing these fundamental dynamics. The analysis reveals that in feed switches or dilution rate shift-ups, the transport enzyme reaches a maximum before RNA achieves its maximum, and in dilution rate shift-downs the cell density reaches a maximum before RNA achieves a minimum.

Citric acid production by selected mutants of Aspergillus niger from cane molasses

The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 ± 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro- N-nitroso-guanidine (MNNG). Out of 3,2-deoxy- d-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 ± 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H 2SO 4 pre-treated blackstrap molasses in Vogel’s medium. On the basis of kinetic parameters such as volumetric substrate uptake rate ( Q s), and specific substrate uptake rate ( q s), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 × 10 −5 M MgSO 4 · 5H 2O into the fermentation medium reduced the Fe 2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6 ± 5 g/l).

Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities.

An integrated study on cell growth, enzyme activities and carbon flux redistribution was made to investigate how the central metabolism of Escherichia coli changes with the knockout of genes in the oxidative pentose phosphate pathway (PPP). Mutants deficient in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were constructed by disrupting the zwf and gnd genes and were grown in minimal media with two different carbon sources, such as glucose or pyruvate. It was shown that the knockout of either gnd or zwf gene did not affect the cell growth rate significantly, but the cellular metabolism was changed. While the specific substrate uptake rate and the specific carbon dioxide evolution rate for either mutant grown on glucose were higher than those obtained for the parent strain, these two rates were markedly decreased in mutants grown on pyruvate. The measurement of enzyme activities implied a significant change in metabolism, when alternative pathways such as the Entner-Doudoroff pathway (EDP) and the malic enzyme pathway were activated in the gnd mutant grown on glucose. As compared with the parent strain, the activities of phosphoglucose isomerase were increased in mutants grown on glucose but decreased in mutants grown on pyruvate. The metabolic flux redistribution obtained based on 13C-labeling experiments further indicated that the direction of the flux through the non-oxidative PPP was reversed in response to the gene knockout. Moreover, the knockout of genes caused an increased flux through the tricarboxlic acid cycle in mutants grown on glucose but caused a decrease in the case of using pyruvate. There was also a negative correlation between the fluxes through malic enzyme and isocitrate dehydrogenase in the mutants; and a positive correlation was found between the fluxes through malic enzyme and phosphoenolpyruvate carboxylase.

Direct production of citric acid from raw starch by Aspergillus niger

The present study deals with the direct production of citric acid from raw starch by Aspergillus niger. Shake flask and semi solid culture methods were compared using A. niger GCB-47 (parental strain) and GCMC-7 (mutant strain). When cultivated in shaking culture with 150 g/l soluble starch as a carbon source, the mutant strain GCMC-7 produced 69.5 g/l citric acid, which was, 1.48-fold greater than the parental strain GCB-47. From a practical viewpoint, direct production of citric acid from corn and potato starch was examined using semi-solid culture. On the basis of a comparison of kinetic parameters namely the volumetric substrate uptake rate (Qs), the specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutant strain was a faster growing organism. The mutant strain GCMC-7 produced 71.4 and 92.9 g/l citric acid, approximately 1.44 and 1.12 times as much as the parental strain GCB-47, from 200 g/l corn and potato starch, respectively. The findings suggest that GCMC-7 possesses enhanced ability for sugar metabolism and citric acid production.

Kinetic analysis of the psychrophilic anaerobic digestion of wastewater derived from the production of proteins from extracted sunflower flour.

A kinetic analysis of the anaerobic digestion process of wastewater derived from the production of protein isolates from extracted sunflower flour was carried out. The digestion was conducted in a laboratory-scale fluidized bed reactor with saponite (magnesium silicate) as support for the mediating bacteria at psychrophilic temperature (15-19 degrees C). Soluble chemical oxygen demand (COD(s)) removal efficiencies in the range of 95.9-69.0% were achieved in the reactor at organic loading rates (OLR) of between 0.57 and 2.49 g total COD (COD(t))/L d, hydraulic retention times (HRT) of between 20.0 and 4.5 days, and average feed total COD concentration of 11.3 g/L. The yield coefficient of methane production was 0.32 L of methane (at STP) per gram of COD(t) removed. The total volatile fatty acid (TVFA) levels and the TVFA/alkalinity ratio were lower than the suggested limits for digester failure for OLR and HRT up to 2.26 g COD(t)/L d and 5.0 days, respectively. The specific rate of substrate uptake, r (g COD(s)/g VSS d), correlated with the concentration of biodegradable substrate, S (g COD(s)/L), through an equation of the Michaelis-Menten type. The maximum substrate utilization rate, k, and the Michaelis constant, K(s)(), were found to be 0.125 g COD(s)/g VSS d and 124 mg COD(s)/L, respectively. This proposed model predicted the behavior of the reactor very accurately showing deviations lower than 10% between the experimental and theoretical values of substrate uptake rates. A mass (COD(t)) balance around the reactor allowed the COD equivalent of methane volume (W(CH)4) to be obtained, which gave a value of 2.89 g COD(t)/L CH(4), which was virtually coincident with the theoretical value of 2.86 g COD(t)/L CH(4).

Quantitative analysis of the impact of HXK2 and REG1 deletion in Saccharomyces cerevisiae on invertase expression and respiration

In Saccharomyces cerevisiae, the genes HXK2 and REG1 are believed to play a central role in carbon catabolite repression, acting on multiple pathways in the central metabolism. The present study aimed at quantifying the effect of deletion of genes HXK2 or REG1, respectively, in the S. cerevisiae strain CEN.PK113-7D on the regulation of invertase and respiratory functions. Batch cultivation of the HXK2 deletion mutant on substrate mixtures of sucrose, fructose and glucose and on pure glucose showed an initial high invertase expression followed by a gradual decrease of specific invertase production as well as respiratory capacity. Our data strongly indicate that this regulatory pattern might be caused by a repression function exerted by the Hxk1 protein. This was concluded from the observation that the HXK2 deletion mutant on pure fructose, for which Hxk1 was the only remaining phosphorylating enzyme, was subjected to a considerably stronger invertase repression than on a fructose/sucrose mixture. The present study confirmed that deletion of HXK2 or REG1 alleviated carbon catabolite repression at any of the tested monosaccharide concentrations. The experiments showed that the deletions affected invertase expression and respiratory functions equally. This suggests that REG1 and HXK2 play a central role in the regulation of carbon catabolic pathways. Finally, we show that the deletion of HXK2 or REG1 enhanced respiratory functions but reduced the specific substrate uptake rate and, as a consequence, biomass productivity. An inverse correlation between oxidative capacity and specific substrate uptake rate was found to hold for all strains and all initial sugar mixtures examined in this work.

Kinetics of chlorophenol degradation by benzoate-induced culture of Rhodococcus erythropolis M1

A pure culture of Rhodococcus erythropolis was isolated with the ability to degrade 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. Degradation of 2-chlorophenol by the uninduced culture of Rhodococcus erythropolis began after a prolonged lag period and complete mineralization of the substrates took 45 days. With the aim of reducing the lag period and subsequently improving the rate of degradation, the cells of the isolate were induced with benzoate, phenol, toluene and catechol individually. Benzoate-induced cells showed the highest rate of degradation and were thus used for the study of the degradation kinetics of 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. Complete mineralization of these substrates was achieved up to a concentration of 300, 100 and 50 mg l−1 respectively. Degradation of the chlorophenols was initiated without any significant lag and took the remarkably short time periods of 84, 64 and 144 h for the highest concentrations of the substrate. Evaluation of kinetic parameters showed chlorophenol degradation to follow substrate inhibition kinetics. This is evident from the decrease in specific growth rate, growth yield and substrate uptake rate with increase in the initial substrate concentrations. Toxicity of the chlorophenols was observed to depend on the position of chlorine on the benzene ring and the degree of chlorination.

Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses

Spore suspensions of Aspergillus niger GCB 75, which produced 31.1 g/l citric acid from 15% sugars in molasses, were subjected to u.v.-induced mutagenesis. Among three variants, GCM 45 was found to be the best citric acid producer and was further improved by chemical mutagenesis using NTG. Out of 3 deoxy-D-glucose-resistant variants, GCM 7 was selected as the best mutant which produced 86.1 ± 1.5 g/l citric acid after 168 h of fermentation of potassium ferricyanide + H2SO4-pretreated black strap molasses (containing 150 g sugars/l) in Vogel's medium. On the basis of comparison of kinetic parameters, namely the volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and had the ability to overproduce citric acid.

Assessment of kinetic and macroenergetic parameters for a mesophilic anaerobic fluidised-bed reactor treating wastewater derived from the production of protein isolates from extracted sunflower flour

A study of the kinetics of the anaerobic digestion process of wastewater derived from the production of protein isolates from extracted sunflower flour was carried out in a laboratory-scale fluidised bed reactor with saponite as support for the mediating bacteria at mesophilic temperature (35°C). The reactor operated satisfactorily between hydraulic retention times (HRT) of 1.1 to 20.0 days, removing between 98.3 and 80.0% of the initial chemical oxygen demand (COD). Macroenergetic parameters, calculated using Guiot's kinetic model, gave a yield coefficient for the biomass ( Y) of 0.075 g VSS/g COD and a specific rate of substrate uptake for cell maintenance ( m) of 0.021 g COD/g VSS day. The experimental results also showed the rate of substrate uptake, R s (g COD/g VSS day) correlated with the concentration of biodegradable substrate, S (g COD/l), through an equation of the Michaelis-Menten type. This proposed model predicted the behaviour of the reactor very accurately showing deviations lower than 10% between the experimental and theoretical values of substrate uptake rates.

Uncoupling of metabolism to reduce biomass production in the activated sludge process

Production of excess biomass during biological treatment of wastewaters requires costly disposal. Also with environmental and legislative constraints limiting disposal options, considerable impetus exists for reducing the amount of biomass produced. Uncoupling metabolism in activated sludge may reduce biomass production and this approach is investigated in conjunction with consequences upon substrate removal and population dynamics. To induce uncoupled metabolism, para-nitrophenol ( pNP) a known protonphoric uncoupler of oxidative phosphorylation, was introduced to a bench-scale activated sludge process. Microbial populations were monitored by both microscopic and by three methods of molecular analyses. Presence of the protonphore caused a shift in the microbial population with protozoa being washed out of the system and filamentous bacteria proliferating. The molecular composition of the microbial community was determined by PCR amplification of 16SrRNA genes and subsequent denaturing gradient gel electrophoresis (DGGE). Band Patterns obtained by both a direct and nested approach were similar. However, profiles derived from nested PCR contained more bands, indicative of the increased sensitivity of this approach. Analysis of the active biomass by Polyacrylamide Gel Electrophoresis (PAGE) of small molecular weight RNA (5mwRNA) showed that a sustained shift in the diversity of the predominant, metabolically active species present occurred within two days of the introduction of the protonphore. Biomass production was reduced by 49%, but the total substrate removal rate was also reduced by 25%. The combined effect was a 30% decrease in the biomass yield. Introduction of the protonphore caused substrate removal efficiency to decrease from a consistent value of 96% to 68.5% with considerable variance. This decline in overall process performance was attributed to a surmountable effect arising from the design of apparatus that resulted in a decrease in the reactor biomass concentration. Although the specific biomass volume was consistent throughout, decreased sedimentation resulted in solids being removed in the final effluent which decreased the amount of biomass which could be recycled. The catalytic efficiency of the biomass increased as reflected by a 3.3 fold increase in the specific substrate uptake rate.

Biodegradation of phenolic compounds by an acclimated activated sludge and isolated bacteria

The degradation of a mixture of phenol, 4-chlorophenol (4CP), 2,4-dichlorophenol (24DCP) and 2,4,6-trichlorophenol (246TCP) by acclimated activated sludge and by isolated bacteria was studied. Activated sludge was acclimated for 70 days to 40 mg phenols/l then the microorganisms responsible for the CP degradation were isolated and identified. Four types of Gram-negative bacteria (Aeromonas sp., Pseudomonas sp. Flavomonas oryzihabitans, and Chryseomonas luteola) were identified. Also, two acid-fast bacilli with distinct glycolipid patterns were isolated. From their chemical composition and their growth characteristics, both isolates appeared to be mycobacteria closely related to Mycobacterium peregrinum. The degradation kinetics of each phenol by Aeromonas sp., Pseudomonas sp. Flavomonas oryzihabitans, Chryseomonas luteola and activated sludge were determined. The acclimated activated sludge degradation rates were from one to two orders of magnitude higher than those of pure strains when uptake rates were calculated in terms of the viable biomass (CFU). The specific substrate uptake rate for acclimated activated sludge varied between 8.2 and 15.8 × 10−7 mg/CFU·d (407-784 mg/gVSS·d). Aeromonas sp. had the highest specific substrate uptake rate of the pure strains, based on a VSS basis (33-57 mg/gVSS·d) but, in terms of viable biomass (5.0-15.6 × 10−8 mg/CFU·d), the Pseudomonas sp. rate was the highest. Specific substrate uptake rates were 1.8 mg chlorinated phenols/g VSS·d for unacclimated activated sludge.

The use of chemical uncouplers for reducing biomass production during biodegradation

Reducing the energy available for anabolism of cell mass was identified as a method to minimise disposal requirements of excess biomass produced in the activated sludge process. A model system consisting of Pseudomonas putida, maintained in a chemostat, was employed to investigate biomass production in the presence of the energy dissipating protonphore, p-nitrophenol (pNP). The efficiency of biomass production was reduced by up to 62% when the feed was supplemented with 100 mg pNP.l−1 with a simultaneous increase in the specific substrate uptake rate. The data obtained have been analysed to reveal maintenance energy requirements and true growth yields. Cells were found to satisfy their maintenance energy requirements prior to utilising energy in anabolism. Decreases in pH alone had no effect on biomass production, but caused additional protonphore induced reduction of biomass production. A pH 6.2 the efficiency of biomass production was reduced by up to 77% when the feed was supplemented with 100 mg pNP.l−1.

Biodegradation of phenolic compounds by an acclimated activated sludge and isolated bacteria

The degradation of a mixture of phenol, 4-chlorophenol (4CP), 2,4-dichlorophenol (24DCP) and 2,4,6-trichlorophenol (246TCP) by acclimated activated sludge and by isolated bacteria was studied. Activated sludge was acclimated for 70 days to 40 mg phenols/1 then the microorganisms responsible for the CP degradation were isolated and identified. Four types of Gram-negative bacteria (Aeromonas sp., Pseudomonas sp. Flavomonas oryzihabitans, and Chryseomonas luteola) were identified. Also, two acid-fast bacilli with distinct glycolipid patterns were isolated. From their chemical composition and their growth characteristics, both isolates appeared to be mycobacteria closely related to Mycobacterium peregrinum. The degradation kinetics of each phenol by Aeromonas sp., Pseudomonas sp. Flavomonas oryzihabitans, Chrvseomonas luteola and activated sludge were determined. The acclimated activated sludge degradation rates were from one to two orders of magnitude higher than those of pure strains when uptake rates were calculated in terms of the viable biomass (CFU). The specific substrate uptake rate for acclimated activated sludge varied between 8.2 and 15.8 ß 10−7 mg/CFU·d (407-784 mg/BVSS·d). Aeromonas sp. had the highest specific substrate uptake rate of the pure strains, based on a VSS basis (33–57 mg/gVSS·d) but, in terms of viable biomass (5.0–15.6 × 10−8 mg/CFU·d), the Pseudomonas sp. rate was the highest. Specific substrate uptake rates were 1.8 mg chlorinated phenoWg VSS-d for unacclimated activated sludge.