Chronic effect of erythromycin on substrate biodegradation kinetics of activated sludge

This study evaluated the chronic impact of erythromycin, a macrolide antibiotic, on microbial activities, mainly focusing on changes in process kinetics induced on substrate biodegradation and all related biochemical processes of microbial metabolism. Experiments involved two fill/draw reactors sustained at steady state at two different sludge ages of 10 and 2.0 days, fed with peptone mixture and continuous erythromycin dosing of 50 mg/L. Oxygen uptake rate profiles were generated in a series of parallel batch reactors seeded with biomass from fill/draw systems at selected periods of steady-state operation. Experimental data were evaluated by model calibration reflecting inhibitory effect on process kinetics: continuous erythromycin dosing inhibited microbial growth, reduced the rate of hydrolysis, blocked substrate storage and accelerated endogenous respiration. Adverse impact was mainly due to changes inflicted on the composition of microbial community. Interruption of erythromycin feeding resulted in partial recovery of microbial response. Sludge age affected the nature of inhibition, indicating different process kinetics for faster growing microbial community. Kinetic evaluation additionally revealed the toxic effect of erythromycin, which inactivated a fraction of biomass. Mass balance using oxygen uptake rate data also identified a stoichiometric impact, where a fraction of available substrate, although completely removed, could not be utilized in metabolic activities.     

Decolorization kinetics of the azo dye Reactive Red 2 under methanogenic conditions: effect of long-term culture acclimation

The biological decolorization of the textile azo dye Reactive Red 2 was investigated using a mixed, mesophilic methanogenic culture, which was developed with mixed liquor obtained from a mesophilic, municipal anaerobic digester and enriched by feeding a mixture of dextrin/peptone as well as media containing salts, trace metals and vitamins. Batch decolorization assays were conducted with the unacclimated methanogenic culture and dye decolorization kinetics were determined as a function of initial dye, biomass, and carbon source concentrations. Dye decolorization was inhibited at initial dye concentrations higher than 100 mg l^-1 and decolorization kinetics were described based on the Haldane model. The effect of long-term culture exposure to the reactive dye on decolorization kinetics, culture acclimation, as well as possible dye mineralization was tested using two reactors fed weekly for two years with an initial dye concentration of 300 mg l^-1 and a mixture of dextrin/peptone. The maximum dye decolorization rate after a 2-year acclimation at an initial dye concentration of 300 mg l^-1 was more than 10-fold higher as compared to that obtained with the unacclimated culture. Aniline and the o-aminohydroxynaphthalene derivative resulting from the reductive azo bond cleavage of the dye were detected, but further transformation(s) leading to dye mineralization were not observed. Reactive Red 2 did not serve as the carbon and energy source for the mixed culture, and dye decolorization was sustained by the continuous addition of dextrin and peptone. Thus, biological decolorization of reactive azo dyes is feasible under conditions of low redox potential created and maintained in overall methanogenic systems, but supply of a biodegradable carbon source is necessary.     

Multisubstrate biodegradation kinetics of naphthalene, phenanthrene, and pyrene mixtures.

Biodegradation kinetics of naphthalene, phenanthrene and pyrene were studied in sole-substrate systems, and in binary and ternary mixtures to examine substrate interactions. The experiments were conducted in aerobic batch aqueous systems inoculated with a mixed culture that had been isolated from soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Monod kinetic parameters and yield coefficients for the individual compounds were estimated from substrate depletion and CO(2) evolution rate data in sole-substrate experiments. In all three binary mixture experiments, biodegradation kinetics were comparable to the sole-substrate kinetics. In the ternary mixture, biodegradation of naphthalene was inhibited and the biodegradation rates of phenanthrene and pyrene were enhanced. A multisubstrate form of the Monod kinetic model was found to adequately predict substrate interactions in the binary and ternary mixtures using only the parameters derived from sole-substrate experiments. Numerical simulations of biomass growth kinetics explain the observed range of behaviors in PAH mixtures. In general, the biodegradation rates of the more degradable and abundant compounds are reduced due to competitive inhibition, but enhanced biodegradation of the more recalcitrant PAHs occurs due to simultaneous biomass growth on multiple substrates. In PAH-contaminated environments, substrate interactions may be very large due to additive effects from the large number of compounds present.     

Microbial dynamics in a continuous stirred tank bioreactor exposed to an alternating sequence of organic compounds

Microbial dynamics during aerobic biodegradation of an alternating mixture of organic compounds was investigated experimentally in a continuous stirred tank bioreactor (CSTB). A mathematical model describing this system was developed and tested using the experimental results. A model microbial culture consisting of Pseudomonas sp. JS150, a monochlorobenzene (MCB) degrader, and Xanthobacter autotrophicus GJ10, a 1,2-dichloroethane (DCE) degrader, each with exclusive degradation capabilities, was used. The CSTB was inoculated with both microbial strains and exposed to an alternating sequence of the two compounds at noninhibitory concentrations. Concentrations of each microbial strain, of each organic compound, and of degradation product evolved, as well as specific microbial activities via oxygen uptake tests, were monitored. Reduction of the residual DCE discharged from the bioreactor after an MCB to DCE transition was successfully achieved by continuously feeding a low flow of a concentrated solution of both compounds.     

Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis

Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.     

Competition between two microbial populations in a sequencing fed-batch reactor: theory, experimental verification, and implications for waste treatment applications

Competition between two microbial populations for a single pollutant (phenol) was studied in a sequencing fed-batch reactor (SFBR). A mathematical model describing this system was developed and tested experimentally. It is based on specific growth rate expressions revealed from pure culture batch experiments. The species employed were Pseudomonas putida (ATCC 17514) and Pseudomonas resinovorans (ATCC 14235). It was found that both species biodegrade phenol following inhibitory kinetics which can be described by Andrews' expression. The model predicts that the dynamics of a SFBR, and the kinetics of biodegradation, result in a complex set of operating regimes in which neither species, only one species, or both species can survive at steady cycle. The model also predicts the existence of multiple outcomes, achievable from different start-up conditions, in some domains of the operating parameter space. Experimental results confirmed the model predictions. There was excellent agreement between predicted and measured concentrations of phenol, total biomass, and the biomass of each individual species. This study shows how serious discrepancies can arise in scale-up of biodegradation data if population dynamics are not taken into account. It also further confirms experimentally the theory of microbial competition in periodically forced bioreactors. (c) 1993 John Wiley & Sons, Inc.     

Biodegradation of phenol and m-cresol in a batch and fed batch operated internal loop airlift bioreactor by indigenous mixed microbial culture predominantly Pseudomonas sp

An internal loop airlift reactor (ILALR) is developed and studied for biodegradation of phenol/m-cresol as single and dual substrate systems under batch and fed batch operation using an indigenous mixed microbial strain, predominantly Pseudomonas sp. The results showed that the culture could degrade phenol/m-cresol completely at a maximum concentration of 600mgl(-1) and 400mgl(-1), respectively. Batch ILALR study has revealed that phenol has been preferentially degraded by the microbial culture rather than m-cresol probably owing to the toxic effect of the later. Sum kinetic model evaluated the interaction between the phenol/m-cresol in dual substrate system, which resulted in a high coefficient of determination (R(2)) value >0.98). The fed batch results showed that the strain was able to degrade phenol/m-cresol with maximum individual concentrations 600mgl(-1) each in 26h and 37h, respectively. Moreover for fed batch operation, degradation rates increased with increase in feed concentration without any lag in the degradation profile.     

Modeling 1,2-dichloroethane biodegradation by <Emphasis Type="Italic">Klebsiella oxytoca va 8391</Emphasis> immobilized on granulated activated carbon

A mathematical model of the biodegradation of xenobiotics by microbial cells attached to particles of granulated activated carbon was developed. The model allowed the quantitative evaluation of different characteristics of the biofilm behavior: retarded microbial growth, increased concentration of immobilized cells compared to suspended cultures, potential cell detachment from the solid support and consequent independent growth of free cells. The applicability of the model was demonstrated for our own experimental data for 1,2- dichloroethane (DCA) biodegradation by Klebsiella oxytoca VA 8391 cells attached to granulated activated carbon. Two types of reactors, recirculated batch and continuous flow bioreactor, were studied. It was shown that in all investigated cases, the major contribution to DCA biodegradation was provided by the immobilized cells. Furthermore, immobilized cells were found to tolerate much higher substrate concentration and dilution rates in continuous culture than the free cells.     

Modeling substrate interactions during the biodegradation of mixtures of toluene and phenol by Burkholderia species JS150

The biodegradation kinetics of toluene, phenol, and a mixture of toluene and phenol by Burkholderia species JS150 was measured and modeled. Both of these compounds can serve as the sole source of carbon and energy for this microorganism. The single-substrate biodegradation kinetics was described well using the Monod model, with model constants of mu(max,T) = 0.39 h(-1) and K(S,T) = 0.011 mM for growth on toluene and mu(max,P) = 0.309 h(-1) and K(S,P) = 0.0054 mM for growth on phenol. Degradation of the mixture of toluene and phenol followed simultaneous utilization kinetics with toluene being the preferred substrate. Toluene was found to inhibit the rate of utilization of phenol while the presence of phenol had little effect on the rate of degradation of toluene. Of the kinetic models that were tested, one developed for microbial degradation of multiple substrates was able to describe substrate interactions and to model the mixture utilization by strain JS150. Simple competitive, noncompetitive, or uncompetitive substrate kinetics were not sufficient to describe the observed inhibitory interactions.     

Biodegradation of 4-chlorophenol by <Emphasis Type="Italic">Arthrobacter chlorophenolicus A6</Emphasis>: effect of culture conditions and degradation kinetics

Among known microbial species, Arthrobacter chlorophenolicus A6 has shown very good potential to treat phenolic wastewaters. In this study, the levels of various culture conditions, namely initial pH, agitation (rpm), temperature (°C), and inoculum age (h) were optimized to enhance 4-chlorophenol (4-CP) biodegradation and the culture specific growth rate. For optimization, central composite design of experiments followed by response surface methodology (RSM) was applied. Results showed that among the four independent variables, i.e., pH, agitation (rpm), temperature (°C), and inoculum age (h) investigated in this study, interaction effect between agitation and inoculum age as well as that between agitation and temperature were significant on both 4-CP biodegradation efficiency and culture specific growth rate. Also, at the RSM optimized settings of 7.5 pH, 207 rpm, 29.6°C and 39.5 h inoculum age, 100% biodegradation of 4-CP at a high initial concentration of 300 mg l⁻¹ was achieved within a short span of 18.5 h of culture. The enhancement in the 4-CP biodegradation efficiency was found to be 23% higher than that obtained at the unoptimized settings of the culture conditions. Results of batch growth kinetics of A. chlorophenolicus A6 for various 4-CP initial concentrations revealed that the culture followed substrate inhibition kinetics. Biokinetic constants involved in the process were estimated by fitting the experimental data to several models available from the literature.     

Design of a large-scale surface-aerated bioreactor for biomass production using a VOC substrate

The design of a large-scale bioreactor for the production of bacterial biomass adapted to the biodegradation of volatile organic compounds was carried out. The bioreactor model used integrated the microbial kinetics and fluid dynamics described by the compartment model approach. The process conditions and kinetic parameters were adopted from the laboratory experimental study of (León, E., Seignez, C., Adler, N., Péringer, P., 1999. Growth inhibition of biomass adapted to the degradation of toluene and xylenes in mixture in a batch reactor with substrates supplied by pulses. Biodegradation 10, 245-250). The performance of the pulsed-batch stirred bioreactor under surface aeration conditions was simulated for different mixing configurations and conditions such as the impeller diameter, number of impellers, stirring speed, and oxygen pressure. The simulations were used for the cost analysis which resulted in the optimal design of the bioreactor.     

Biodegradation of phenol and <Emphasis Type="Italic">m</Emphasis>-cresol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 under anaerobic condition

Strain Candida albicans PDY-07 was used to study the anaerobic biodegradation of phenol and m-cresol as single and dual substrates in batch cultures. The strain had a higher potential to degrade phenol than m-cresol. The cell growth kinetics of batch cultures with various initial m-cresol concentrations was investigated, and the Haldane kinetic model adequately described the dynamic behavior of cell growth on m-cresol. When cells grew on the mixture of phenol and m-cresol, substrate interactions were observed. Phenol inhibited the utilization of m-cresol; on the other hand, m-cresol also inhibited the degradation of phenol. However, the presence of low-concentration phenol enhanced m-cresol biodegradation; 100 mg/l m-cresol could be completely degraded within a shorter period of time than m-cresol alone in the presence of 150–300 mg/l phenol. The maximum m-cresol biodegradation rate was obtained at the existence of 200 mg/l phenol. Phenol was preferably utilized by the strain as a carbon and energy source. In addition, a sum kinetics model was used to describe the cell growth behavior in binary mixture of phenol and m-cresol, and the interaction parameters were determined. The model adequately predicted the growth kinetics and the interaction between the substrates.     

Improved production of an enzyme that hydrolyses raw yam starch by Penicillium sp. S-22 using fed-batch fermentation

A newly isolated strain, Penicillium sp. S-22, was used to produce an enzyme that hydrolyses raw yam starch [raw yam starch digesting enzyme (RYSDE)]. The enzyme activity and overall enzyme productivity were respectively 16 U/ml and 0.19 U/ml h in the batch culture. The enzyme activity increased to 85 U/ml by feeding of partially hydrolyzed raw yam starch. When a mixture containing partially hydrolyzed raw yam starch and peptone was fed by a pH-stat strategy, the enzyme activity reached 366 U/ml, 23-fold of that obtained in the batch culture, and the overall productivity reached 3.4 U/ml h, which was 18-fold of that in the batch culture.     

Influence of Primatone RL supplementation on sialylation of recombinant human interferon-gamma produced by Chinese hamster ovary cell culture using serum-free media

Although serum-free media have been widely used in mammalian cell culture for therapeutic protein production, the effects of serum-substitutes on product quality have not been extensively examined. This study observed an adverse effect of Primatone RL, an animal tissue hydrolysate commonly used as a serum-substitute to promote cell growth, on sialylation of interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture in both batch and fed-batch modes. In batch cultures, decreased sialylation was observed at each of the glycosylation sites (i.e., Asn(25) and Asn(97)) of IFN-gamma with the use of elevated concentrations of the peptone. Although poorest sialylation was obtained with the use of a growth-inhibiting concentration of Primatone RL, diminished sialylation was observed at the optimal peptone concentration for cell growth and product yield. Since incubation of the product in Primatone RL-supplemented acellular medium did not result in decreased sialylation, the negative effect of Primatone RL could not be attributed to extracellular desialylation of IFN-gamma by components of the peptone. In the fed-batch mode, a culture utilizing a serum-free feeding medium supplemented with Primatone RL demonstrated poorer sialylation than a similar culture not fed the peptone. The results of both the batch and fed-batch experiments indicate that the adverse effect of the peptone was not due solely to ammonia accumulation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 353-360, 1997.     

Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied with the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.     

Toward a stochastic formulation of microbial growth in relation to bioreactor performances: case study of an E. coli fed-batch process

A stochastic microbial growth model has been elaborated in the case of the culture of E. coli in fed-batch and scale-down reactors. This model is based on the stochastic determination of the generation time of the microbial cells. The determination of generation time is determined by choosing the appropriate value on a log-normal distribution. The appropriateness of such distribution is discussed and growth curves are obtained that show good agreement compared with the experimental results. The mean and the standard deviation of the log-normal distribution can be considered to be constant during the batch phase of the culture, but they vary when the fed-batch mode is started. It has been shown that the parameters related to the log-normal distribution are submitted to an exponential evolution. The aim of this study is to explore the bioreactor hydrodynamic effect on microbial growth. Thus, in a second time, the stochastic growth model has been reinforced by data coming from a previous stochastic bioreactor mixing model (1). The connection of these hydrodynamic data with the actual stochastic growth model has allowed us to explain the scale-down effect associated with the glucose concentration fluctuations. It is important to point out that the scale-down effect is induced differently according to the feeding strategy involved in the fed-batch experiments.     

Batch kinetics of microbial polysaccharide biosynthesis

A modified form of logistic equation has been proposed to quantity the batch kinetics of microbial growth during the biosynthesis of extra- and intracellular polymers. Based on the experimental data developed in this study, the proposed model appeared to provide adequate growth and fermentation kinetics of Aureobasidium pullulans. The model was also applicable for representing the reported data on pullulan, xanthan, and poly-beta-hydroxybutyricacid. In comparison to the logistic and Monod kinetics, this model fitted the data better and more accurately described the overall fermentation, both concentrations and fermentation time.     

Kinetics of biodegradation of gasoline and its hydrocarbon constituents

Aerobic biodegradation of gasoline and its constituents, benzene, toluene and ethylbenzene were studied by an enrichment from soil indigenous microbial population. The enrichment culture completely degraded 16.1–660 mg/l gasoline in 2.5–16 days respectively, without accumulation of any by-products. The kinetics of gasoline as well as benzene, toluene and ethylbenzene biodegradation was investigated with initial gasoline concentrations of 16.1–62.6 mg/l. The maximum specific rates of biodegradation of benzene, toluene and ethylbenzene were 0.12, 0.38 and 0.19 mg mg biomass⁻¹ day⁻¹ respectively. When benzene and toluene were used as sole substrate, the maximum specific rates of their biodegradation were 62.9 and 16.4 times greater than the corresponding values for a mixture (gasoline). The microbial culture was able to mineralize up to 200 mg/l pure toluene and benzene. Maximum mineralization efficiencies of benzene and toluene were 76.7 ± 5.1% and 76.8 ± 1.3% respectively. Self-inhibition and competitive inhibition patterns were observed during the biodegradation of benzene and toluene alone and in the mixture respectively. The observed kinetics was modeled according to Andrews' inhibition model. Received: 6 August 1997 / Received revision: 18 November 1997 / Accepted: 29 November 1997