Kinetics of microbial growth on pentachlorophenol

Batch and fed-batch experiments were conducted to examine the kinetics of pentachlorophenol utilization by an enrichment culture of pentachlorophenol-degrading bacteria. The Haldane modification of the Monod equation was found to describe the relationship between the specific growth rate and substrate concentration. Analysis of the kinetic parameters indicated that the maximum specific growth rate and yield coefficients are low, with values of 0.074 h-1 and 0.136 g/g, respectively. The Monod constant (Ks) was estimated to be 60 micrograms/liter, indicating a high affinity of the microorganisms for the substrate. However, high concentrations (KI = 1,375 micrograms/liter) were shown to be inhibitory for metabolism and growth. These kinetic parameters can be used to define the optimal conditions for the removal of pentachlorophenol in biological treatment systems.     

Kinetics of microbial growth on pentachlorophenol.

Batch and fed-batch experiments were conducted to examine the kinetics of pentachlorophenol utilization by an enrichment culture of pentachlorophenol-degrading bacteria. The Haldane modification of the Monod equation was found to describe the relationship between the specific growth rate and substrate concentration. Analysis of the kinetic parameters indicated that the maximum specific growth rate and yield coefficients are low, with values of 0.074 h-1 and 0.136 g/g, respectively. The Monod constant (Ks) was estimated to be 60 micrograms/liter, indicating a high affinity of the microorganisms for the substrate. However, high concentrations (KI = 1,375 micrograms/liter) were shown to be inhibitory for metabolism and growth. These kinetic parameters can be used to define the optimal conditions for the removal of pentachlorophenol in biological treatment systems.     

Nutrient-limited microbial growth kinetics: overview and recent advances

Traditional concepts of nutrient uptake and growth kinetics as linked by cell yield are presented. Phenomena affecting the kinetics are examined along with a discussion of those which lead to ambiguity. Concepts of flux control are presented to help understand the distribution of material along metabolic pathways. Specific affinity is described to relate nutrient accumulation rates to transporter density. It is shown to be a primary kinetic constant and the best available index of nutrient collection ability. As an aid to understanding, specific affinity is reexpressed in terms of membrane permeability. Formulations of nutrient transport rate as a function of cellular composition, particularly transporter and enzyme content and known as janusian kinetics, are described as an improvement to specific affinity theory. Procedures for quantified unidirectional fluxes are reviewed to identify the difference between gross and net transport rates of substrate. Collision frequency theory is used to show that in addition to total biomass, cell size and transporter density should also be included in rate equations describing microbial growth. Theory diversity suggests that one reason for microbial metabolic is that the likelihood of additional collisions of substrate molecules with a cell surface, after an initial collision, requires only a sparse distribution of transporter sites for maximal rate, leaving room for additional transporters able to collect other substrate types.     

Simultaneous Rates of Ribonucleic Acid and Deoxyribonucleic Acid Syntheses for Estimating Growth and Cell Division of Aquatic Microbial Communities

A method for measuring rates of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) syntheses using a single radioactive precursor has been devised and tested using bacterial cultures and natural assemblages of marine and freshwater microorganisms. The procedure is based upon the uptake and incorporation of exogenous [³H]adenine into cellular adenosine triphosphate and deoxyadenosine triphosphate pools which serve as the immediate precursors for the adenine incorporated into RNA and DNA, respectively. It is proposed that the DNA/RNA rate ratio is correlated with the specific growth rate of microorganisms and can be used as an index for estimating and comparing the productivities of microbial assemblages in nature. This technique can also be used to detect discontinuous growth and cell division processes which frequently occur in surface plankton populations. The DNA/RNA rate ratios measured in a variety of aquatic ecosystems ranged from 3.3 to 31.8% without significant correlation to total microbial biomass.     

Attached and Free-Floating Bacteria in a Diverse Selection of Water Bodies

The contribution of attached and free-floating bacteria to the bacterial numbers and heterotrophic uptake in 44 diverse aquatic environments was studied. A factor analysis reduced the variability of the raw data base to three major factors explaining 53.6% of total variance. These factors were (i) salinity, (ii) heterotrophic uptake, and (iii) particulate load. A cluster analysis categorized the 44 habitats into five distinct environmental types based on these three factors. There was no significant pattern in the distribution of attached versus free-floating bacteria when assessed by epifluorescent microscopy. However the contribution of attached bacteria to the uptake of an amino acids mix was reduced in marine waters. Heavy particulate loads resulted in an increased percentage uptake of amino acids and glucose from the attached bacteria. Uptake response was found to be substrate specific especially in oliogotrophic freshwater. Amino acid uptake was more associated with the attached fraction, whereas glucose uptake was mediated more by the free-floating fraction.     

``BACWAVE,' a Spatial–Temporal Model for Traveling Waves of Bacterial Populations in Response to a Moving Carbon Source in Soil

Abstract Previously, we discovered the phenomenon of wavelike spatial distributions of bacterial populations and total organic carbon (TOC) along wheat roots. We hypothesized that the principal mechanism underlying this phenomenon is a cycle of growth, death, autolysis, and regrowth of bacteria in response to a moving substrate source (root tip). The aims of this research were (i) to create a simulation model describing wavelike patterns of microbial populations in the rhizosphere, and (ii) to investigate by simulation the conditions leading to these patterns. After transformation of observed spatial data to presumed temporal data based on root growth rates, a simulation model was constructed with the Runge–Kutta integration method to simulate the dynamics of colony-forming bacterial biomass, with growth and death rates depending on substrate content so that the rate curves crossed over at a substrate concentration within the range of substrate availability in the model. This model was named ``BACWAVE,' standing for ``bacterial waves.' Cyclic dynamics of bacteria were generated by the model that were translated into traveling spatial waves along a moving nutrient source. Parameter values were estimated from calculated initial substrate concentrations and observed microbial distributions along wheat roots by an iterative optimization method. The kinetic parameter estimates fell in the range of values reported in the literature. Calculated microbial biomass values produced spatial fluctuations similar to those obtained for experimental biomass data derived from colony forming units. Concentrations of readily utilizable substrate calculated from biomass dynamics did not mimic measured concentrations of TOC, which consist not only of substrate but also various polymers and humic acids. In conclusion, a moving pulse of nutrients resulting in cycles of growth and death of microorganisms can indeed explain the observed phenomenon of moving microbial waves along roots. This is the first report of wavelike dynamics of microorganisms in soil along a root resulting from the interaction of a single organism group with its substrate. Received: 2 October 1999; Accepted: 9 March 2000; Online Publication: 28 August 2000     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water.

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

The effect of substrate shock on populations of starving aquatic bacteria

The effect of substrate (glucose) shock on starved mixed cultures of aquatic bacteria under conditions resembling the natural aquatic situation has been studied. The prevalent organism in the system was a Pseudomonas sp., and short-term loss of viability and long-term loss of several biochemical properties of this strain were observed. Glucose uptake rate and cell size of the population increased immediately after the shock and synchronous growth occurred subsequently. The population which became established after the shock exhibited lower glucose uptake rates at low substrate concentrations than the starved population. The relevance to similar phenomena which occur when aquatic bacteria are transferred to cultivation media is discussed.     

Specific growth rates of natural microbial communities measured by adenine nucleotide pool turnover

A novel experimental approach has been development to estimate the mean specific growth rates of natural populations of microorganisms. The method is based on the direct measurement of the turnover rate of the total adenine nucleotide (AN)pool using either [³H]adenine or ³²PO₄ radiotraces. In laboratory cultures of bacteria and unicellular algae, the AN pool turnover rate is highly correlated with specific growth rate. This empirically derived relationship is then used to extrapolate AN pool turnover rates measured in the field to population-specific growth rates. The method is technically simple, straightforward and potentially capable of measuring a wide range of growth rates. Microbial community growth rate estimates measured using this new method ranged from 0.04 to 2.45 per day for samples collected from a variety of aquatic habitats. Growth rate estimates derived from data on AN pooll turnover compared favorably with estimates derived from measurements of microbial biomass and production. The AN pool turnover methos has numerous potential applications in applied and environmental microbiology.     

Antagonism between Bacteria and Fungi on Decomposing Aquatic Plant Litter

Bacterial and fungal decomposers of aquatic plant litter may exhibit either synergistic or antagonistic interactions, which are likely to influence microbial growth as well as the decomposition of litter and, eventually, the carbon metabolism of aquatic systems. To elucidate such interactions, we inoculated decomposing Phragmites culms in microcosms with fungal isolates and with natural communities of bacteria and fungi in different combinations. The development of fungal and bacterial biomass and the carbon dynamics were studied during several months of degradation. The results show a bilateral antagonistic relationship between bacteria and fungi. After 3 months, fungal biomass accumulation was approximately 12 times higher in the absence than in the presence of bacteria. Bacterial biomass accumulation was about double in the absence of fungi compared to when fungi were present. Similar interactions developed between a natural assemblage of bacteria and five different fungal strains isolated from Phragmites litter (three identified hyphomycetes and two unidentified strains). Despite the great difference in biomass development between the treatments, the carbon metabolism was similar regardless of whether fungi and/or bacteria were present alone or in coexistence. We suggest that the antagonism between bacteria and fungi is an important controlling factor for microbial colonization and growth on aquatic plant litter.     

Distinctive microbial ecology and biokinetics of autotrophic ammonia and nitrite oxidation in a partial nitrification bioreactor

Biological nitrogen removal (BNR) based on partial nitrification and denitrification via nitrite is a cost-effective alternate to conventional nitrification and denitrification (via nitrate). The goal of this study was to investigate the microbial ecology, biokinetics, and stability of partial nitrification. Stable long-term partial nitrification resulting in 82.1 +/- 17.2% ammonia oxidation, primarily to nitrite (77.3 +/- 19.5% of the ammonia oxidized) was achieved in a lab-scale bioreactor by operation at a pH, dissolved oxygen and solids retention time of 7.5 +/- 0.1, 1.54 +/- 0.87 mg O(2)/L, and 3.0 days, respectively. Bioreactor ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) populations were most closely related to Nitrosomonas europaea and Nitrobacter spp., respectively. The AOB population fraction varied in the range 61 +/- 45% and was much higher than the NOB fraction, 0.71 +/- 1.1%. Using direct measures of bacterial concentrations in conjunction with independent activity measures and mass balances, the maximum specific growth rate (micro(max)), specific decay (b) and observed biomass yield coefficients (Y(obs)) for AOB were 1.08 +/- 1.03 day(-1), 0.32 +/- 0.34 day(-1), and 0.15 +/- 0.06 mg biomass COD/mg N oxidized, respectively. Corresponding micro(max), b, and Y(obs) values for NOB were 2.6 +/- 2.05 day(-1), 1.7 +/- 1.9 day(-1), and 0.04 +/- 0.02 mg biomass COD/mg N oxidized, respectively. The results of this study demonstrate that the highly selective partial nitrification operating conditions enriched for a narrow diversity of rapidly growing AOB and NOB populations unlike conventional BNR reactors, which host a broader diversity of nitrifying bacteria. Further, direct measures of microbial abundance enabled not only elucidation of mixed community microbial ecology but also estimation of key engineering parameters describing bioreactor systems supporting these communities.     

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.     

Effect of antecedent growth conditions on sensitivity of Escherichia coli to chlorine dioxide.

Bacterial resistance to inactivation by antibacterial agents that is induced by the growth environment was studied. Escherichia coli was grown in batch culture and in a chemostat, and the following parameters were varied: type of substrate, growth rate, temperature, and cell density during growth. Low doses (0.75 mg/liter) of chlorine dioxide were used to inactivate the cultures. The results demonstrated that populations grown under conditions that more closely approximated natural aquatic environments were more resistant than those grown under commonly employed batch culture conditions. In particular, bacteria grown at submaximal rates were more resistant than their counterparts grown at mumax. The most resistant populations encountered in this study were those grown at D values of 0.02 h-1 and 0.06 h-1 at 25 degrees C. Growth at 15 degrees C led to greater resistance than did growth at 37 degrees C. The conditions that produced relatively resistant phenotypes were much closer to those found in most natural environments than are the typical conditions of batch culture methods. The importance of major physiological changes that can be induced by the antecedent growth environment is discussed in light of the possible modes of action of several disinfectants.     

Batch Production of Protein from Ethane and Ethane-Methane Mixtures

A culture of Graphium grows upon natural gas and a mineral salt solution. Ethane is the preferred substrate but methane is co-utilized. A stirred-tank type fermentor was used to study batch growth. Maximum production rate of biomass was 80 mg/liter.hr, at pH 4, using simple synthetic supporting medium with ammonium sulfate as a nitrogen source. This rate was observed after 40 hr of fermentation. A doubling time of 3.7 hr was observed. The corresponding specific growth rate was 0.187 per hr. A magnetic drive fermentor was used to study the effect of continuous recycle of gases in a gas-tight system. The rate of oxygen utilization is approximately 2.1 times higher than for ethane. Oxygen must not be allowed to become limiting in recycle gases. The calculated efficiency of overall biomass synthesis averages 30%. Hyphal and unicellular tissue of Graphium contains 52% protein. It compares favorably with standard FAO protein in its content of amino acids.     

Predation-mediated shifts in size distribution of microbial biomass and activity during detritus decomposition

Many experimental studies on detritus decomposition revealed a comparable microbial succession after the addition of a substrate pulse: from small, freely suspended single bacteria at the beginning, to more complex and larger growth forms during a later stage, accompanied by the appearance of bacterivorous protists. We examined in three model experiments with different organic carbon sources whether this shift in bacterial size structure is linked to the grazing impact of bacterivores. In short‐term (8–10 d) microcosm experiments we added natural dissolved and particulate detritus (macrophyte leaves and leachate, dead phytoplankton cells) as an organic substrate source. By the use of size‐fractionated inocula and eucaryotic inhibitors we obtained treatments without protists, in which bacteria developed without predation. These were compared, by measurements of bacterial activity and microscopical analysis of bacterial size structure, to incubations in which either cultured heterotrophic nanoflagellates or a natural protist assemblage was included in the inoculum. The presence of bacterial grazers resulted in a 50–90% reduction of bacterial biomass compared to grazer‐free trials. The selective removal of freely suspended bacteria produced a very different relative composition of bacterial biomass: it became dominated by large, grazing‐resistant forms such as filaments and cells attached to particles or clustered in small aggregates. In grazer‐free treatments, bacterial biomass was always dominated (>80%) by free‐living, single bacterial cells. The time course of the bacterial development suggested different underlying mechanisms for the appearance of predation resistant filamentous and of aggregated or attached bacteria. As bacterial aggregates developed in approximately similar amounts with and without grazers no specific growth stimulation by protists could be detected. In contrast, concentrations of filamentous bacteria were 2–10 times higher in treatments with protists, thus indicating a stimulation of this growth form during enhanced grazing pressure. Measurements of ectoenzymatic activity and H‐leucine uptake indicated that microbial activity was also shifted to larger size fractions. In most cases more than 50% of bacterial activity in treatments with protists was associated with the size fraction>10 μm whereas this value was <2% without grazers. Grazing by protists also enhanced the specific activity of the bacterial assemblage which is in contrast to an assumed lower competitive ability of complex bacterial growth forms. The results imply that the selective force of bacterivory in nutrient‐rich environments changes the structure and possibly the function of aquatic bacteria and their position in the food web, making protist‐resistant bacteria more vulnerable to metazoan filter feeders and detritivores, and possibly also subject to sedimentation.     

Plots of turnover times versus added substrate concentrations provide only upper bounds to in situ substrate concentrations

In recent years it has become a common practice in the study of microbial activity in aquatic systems to estimate in situ substrate concentrations by plotting substrate turnover times (the substrate concentration divided by microbial uptake rate) against the concentrations of added radioactively labeled substrate. The rationale has been that the uptake rate will remain essentially constant if added concentrations of high specific activity substrates are small compared to in situ concentrations. Unfortunately, a correct mathematical analysis shows that the in situ concentrations estimated by this method are only upper bounds to the true in situ substrate concentrations, regardless of the size of the substrate spikes. The estimated in situ concentration will equal the true in situ concentration only if uptake rate is completely independent of substrate concentration, an unlikely situation in natural systems.     

Effects of inhibition and repression on the utilization of substrates by heterogeneous bacterial communities

This investigation attempts to evaluate to what extent enzyme inhibition and repression by metabolites, indigenous to the cell, are significant phenomena in natural microbial communities. Three case histories of the kinetics of substrate utilization and growth in multisubstrate media by heterogeneous bacterial populations are presented: (i) concurrent substrate utilization and growth on both substrates simultaneously (glucose plus benzoate); (ii) sequential substrate elimination accompanied by diauxic growth as a result of inhibition of enzyme activity (glucose plus galactose); (iii) sequential substrate utilization accompanied by diauxic growth caused by repression of enzyme formation (glucose plus l-phenylalanine, benzoate plus l-phenylalanine). It is shown that enzyme inhibition was observed in two-substrate media as well as in multisubstrate media and was maintained at low substrate concentrations (few milligrams per liter). A special attempt has been made to maintain the diversity of the experimental microbial population during the adaptation and enrichment period. All substrates were determined with sensitive analytical methods specific for the individual substrates. The results obtained confirm that catabolite repression and the resulting sequential substrate utilization are observed in heterogeneous bacterial populations.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.