Multisubstrate System Research Articles

Assessing energy fluxes and carbon use in soil as controlled by microbial activity - A thermodynamic perspective A perspective paper

Soil organic matter (SOM) plays a central role for both the C cycle and soil functions. Plants provide the input and heterotrophic (micro)organisms are essential for the turnover. Microbial metabolism links matter and energy fluxes and generates the highest energy turnover dynamics in SOM because the organisms need both energy and matter for maintenance and growth. In this perspectives paper, we evaluate the knowledge on thermodynamic approaches potentially applicable to study the turnover of organic matter in the soil system. Thermodynamics is essential for understanding organic matter turnover in soil as turnover and storage are controlled by the energy supply to, and consumption by, microbes. Instead of just comparing the heat of combustion of compounds without considering microbial anabolism, we need to apply conventional thermodynamic state variables that can be either estimated using established thermodynamic equations, or measured empirically in soil. In particular, we can follow and quantify overall changes of enthalpies by calorimetry. Here, we suggest to apply a thermodynamic concept with the related experimental approaches of calo(respiro)metry and turnover mass balances including biomass formation. This enables us to better interpret and understand the highly variable carbon use efficiency (CUE) in a multi-substrate system such as soil and to relate this to energy use efficiency (EUE). Combining the experimental measurements of the thermodynamic state variables with mass turnover data allow prediction of whether compounds can be metabolized with energy delivery to microorganisms, or be thermodynamically stabilized under the respective redox and electron acceptor conditions. Energy balancing shows how much energy is actually used and retained in the soil, how much is emitted as heat, and how much may be stabilized due to endergonic turnover reactions. Thermodynamic stabilization should therefore be considered as basic stabilization process for organic compounds in soil.

Variation in cell surface characteristics and extracellular polymeric substances during the biodegradation of monocyclic and heterocyclic aromatic hydrocarbons in single and multi-substrate systems

ABSTRACTThe variation in surface characteristics and the composition of extracellular polymeric substances (EPS) of bacterial cells during biodegradation of single and multi-aromatic hydrocarbons was investigated in the present study. The maximum cell surface hydrophobicity (CSH) of 80.1% was observed during the degradation of toluene. Bacterial cells acquired more negative surface charge with an increase in CSH and vice versa. Proteins constituted the major fraction of EPS during biodegradation of benzene and toluene with protein/carbohydrate ratio varying between 2.19 and 3.1. Carbohydrates constituted the major fraction of EPS in the presence of pyridine. A significant variation in cell surface characteristics was observed in multi-substrate systems involving heterocyclic and monocyclic aromatic hydrocarbons. An increase in EPS production (62.89 mg/g) did not facilitate enhanced degradation of hydrophobic substrates in multi-substrate system involving benzothiophene, benzofuran, benzene and toluene. Under toxic conditions, especially at higher concentration of target pollutants, a significant increase in concentration of polysaccharides was observed compared to proteins.

Variation in toxicity during the biodegradation of various heterocyclic and homocyclic aromatic hydrocarbons in single and multi-substrate systems

In the present study, an attempt was made to understand the variation in the toxicity during the biodegradation of aromatic hydrocarbons in single and multi-substrate system. The bacterial bioassay based on the inhibition of dehydrogenase enzyme activity of two different bacterial sp. E.coli and Pseudomonas fluorescens was used for toxicity assessment. Amongst the chosen pollutants, the highest acute toxicity was observed for benzothiophene followed by benzofuran having EC50 value of 16.60mg/L and 19.30mg/L respectively. Maximum residual toxicity of 30.8% was observed at the end during the degradation of benzothiophene. Due to the accumulation of transitory metabolites in both single and multisubstrate systems, reduction in toxicity was not proportional to the decrease in pollutant concentration. In multi-substrate system involving mixture of heterocyclic hydrocarbons, maximum residual toxicity of 39.5% was observed at the end of biodegradation. Enhanced degradation of benzofuran, benzothiophene and their metabolic intermediates were observed in the presence of naphthalene resulting in significant reduction in residual toxicity. 2 (1H) - quinolinone, an intermediate metabolite of quinoline was observed having significant eco-toxicity amongst all other intermediates investigated.

Bioremediation of Polycyclic Aromatic Hydrocarbon Contaminated Soil by a Microbial Consortium through Supplementation of Biosurfactant Produced by Pseudomonas aeruginosa Strain

Lipopeptide biosurfactant produced by Pseudomonas aeruginosa Lbp 3 strain isolated from petroleum contaminated soil was investigated for its potential to enhance bioavailability, and hence, the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil microcosms. Experiments were conducted on a soil spiked with equal parts of the PAHs Phenanthrene, Fluoranthene, and Pyrene to a final concentration of 1200 mg of total PAHs per kg of dry soil. To evaluate biodegradation enhancement efficiency, 50 g spiked soil samples were supplemented with 50 mgL−1, 100 mgL−1, 300 mgL−1, and 1000 mgL−1of lipopeptide dissolved in 30 mL of MSM, and incubated for 40 days at 30°C in darkness. Statistically significant (P < 0.05) biodegradation rates were observed in all the amended microcosms in comparison to the unamended controls. Maximal biodegradations (> 96% of Phenanthrene and Fluoranthene and > 93% of Pyrene) were observed in the soil microcosms supplemented with 1000 mgL−1and 50 mgL−1 lipopeptide. The effect of substrate interactivity of the PAHs on the biodegradation kinetics was also tested in comparison with sole substrate microcosms. Competitive inhibition of the biodegradation of low molecular weight PAHs was observed as a result of substrate interactivity in the multisubstrate system.

Main and Interactive Effects of Polyaromatic Sulfur Heterocyclic Compounds on Growth and Biodegradation Efficiencies of Bacillus sphaericus HN1: Modeling and Statistical Analysis

Bioremediation is an effective technique to remediate soil and water contaminated with a mixture of recalcitrant polyaromatic sulfur heterocyclic (PASH) compounds. Based on two levels of full factorial design, a statistical design of experiments was used in this study to investigate the interactive effects of three PASHs; thiophene (Th), benzothiophene (BT), and dibenzothiophene (DBT) on their biodegradation in multisubstrate batch process using Bacillus sphaericus HN1. Kinetic modeling was performed to estimate the rate of biodegradation of each PASH in single-, binary-, and tertiary-substrate systems. Biodegradation of BT, DBT, and total PASHs follows the first order kinetic reaction in the single- and multisubstrate batch systems, while biodegradation of Th follows the first-order kinetic reaction, in single-substrate system, but second-order kinetic reaction in multisubstrate batch system. Four linear interaction regression models were estimated and validated to describe the interactive effect of the three PASHs on their biodegradation. Statistical analysis of the results in the form of analysis of variance and Student t test indicated significant role played by the main effects of Th, BT and DBT on their biodegradation (p < 0.01). Th and the interactive effect between Th and DBT has statistically significant negative effect on DBT removal (p = 0.0665). However, DBT expresses higher statistically significant positive effect on total PASHs removal than that of Th (p = 0.0178 and p = 0.026, respectively), but their interaction has a significantly negative effect (p = 0.0143).

PAHs biodegradation potential of indigenous consortia from agricultural soil and contaminated soil in two-liquid-phase bioreactor (TLPB)

Estimation of PAHs degradation potential of indigenous consortia is essential for remediation of polluted soils. In this study, the biodegradation of a mixture of 11 PAHs was compared using a long-term PAH-contaminated soil (CS) and an unpolluted agricultural soil (AS) as inocula in a two-liquid-phase bioreactor (TLPB). In the TLPB, silicone oil was used as the organic phase to increase the PAHs bioavailability. The microbial numbers were also determined during the biodegradation. The results demonstrated that naphthalene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene could be completely biodegraded in both soils within 4–50 days. With the exception of dibenzo(a,h)anthrancene, the other PAHs including benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene and benzo(k)fluoranthene were degraded to different extents in both soils at the end of 170 days. Complete biodegradation of benzo(a)anthracene and benzo(b)fluoranthene only occurred in CS. During the process, microbial growth was highly correlated to the biodegradation of PAHs. Sequential utilization of PAHs showed a competitive-inhibition in the multi-substrate system. The half-life times of PAHs obtained here were much shorter than those reported previously in soils, indicating that indigenous microbes in both soils had high PAHs degradation potential, facilitated by TLPB.

Kinetics of growth and multi substrate degradation by an indigenous mixed microbial culture isolated from a wastewater treatment plant in Guwahati, India

An indigenous mixed culture of microorganisms, isolated from a sewage treatment plant, was investigated for its potential to simultaneously degrade phenol and m-cresol during its growth in batch shake flasks. 2(2) full factorial designs with the two substrates as the factors, at two different levels and two different initial concentration ranges, were employed to carry out the biodegradation experiments. For complete utilisation of phenol and m-cresol, the culture took a minimum duration of 21 hrs at their low concentration of 100 mg/L each, and a maximum duration of 187 hrs at high concentration of 600 mg/L each in the multisubstrate system. The biodegradation results also showed that the presence of phenol in low concentration range (100-300 mg/L did not inhibit m-cresol biodegradation; on the other hand, presence of m-cresol inhibited phenol biodegradation by the culture. Moreover, irrespective of the concentrations used, phenol was degraded preferentially and earlier than m-cresol. During the culture growth, a lag phase was observed above a combined concentration of 500 mg/L i.e., 200 mg/L m-cresol and 300 mg/L of phenol and above). Statistical analysis of the specific growth rate of the culture in the multisubstrate system was also performed in the form of ANOVA and Student 't' test, which gave good interpretation in terms of main and interaction effects of the substrates.

Potential rates of fermentation in digesta from the gastrointestinal tract of pigs: effect of feeding fermented liquid feed.

Microbial catabolic capacity in digesta from the gastrointestinal tract of pigs fed either dry feed or fermented liquid feed (FLF) was determined with the PhenePlate multisubstrate system. The in vitro technique was modified to analyze the kinetics of substrate catabolism mediated by the standing stock of enzymes (potential rates of fermentation), allowing a quantitative evaluation of the dietary effect on the catabolic capacity of the microbiota. In total, the potential rates of fermentation were significantly reduced in digesta from the large intestine (cecum, P < 0.1; colon, P < 0.01; and rectum, P < 0.0001) of pigs fed FLF compared to pigs fed dry feed. No effect of diet was observed in the stomach (P = 0.71) or the distal part of the small intestine (P = 0.97). The highest rates of fermentation and the most significant effect of diet were observed for readily fermentable carbohydrates like maltose, sucrose, and lactose. Feeding FLF to pigs also led to a reduction in the large intestine of the total counts of anaerobic bacteria in general and lactic acid bacteria specifically, as well as of microbial activity, as determined by the concentration of ATP and short-chain fatty acids. The low-molecular-weight carbohydrates were fermented mainly to lactic acid in the FLF before being fed to the animals. This may have limited microbial nutrient availability in the digesta reaching the large intestine of pigs fed FLF and may have caused the observed reduction in activity and density of the cecal and colonic microbial population. On the other hand, feeding FLF to pigs reduced the viable counts of coliform bacteria (indicator of Escherichia coli and Salmonella spp.) most profoundly in the stomach and the distal part of the small intestine, probably due to the bactericidal effect of lactic acid and low pH. The results presented clearly demonstrate that feeding FLF to pigs had a great impact on the indigenous microbiota, as reflected in bacterial numbers, short-chain fatty acid concentration, and substrate utilization. However, completely different mechanisms may be involved in the proximal and the distal parts of the gastrointestinal tract. The present study illustrates the utility of the PhenePlate system for quantifying the catabolic capacity of the indigenous gastrointestinal tract microbiota.

Analysis of oxygenation reactions in a multi‐substrate system—A new approach for estimating substrate‐specific true yields

A series of experiments was performed in an aerobic chemostat reactor using a multi-substrate system consisting of acetate, phenol, and 2,4-dichlorophenol (DCP). The phenolic compounds require initial oxygenation reactions, while acetate is oxidized without oxygenations. The biomass completely dechlorinated DCP and utilized all of the substrates simultaneously as electron donors and carbon sources. However, DCP removal was less than for phenol and depended on the solids retention time. A novel substrate-specific yield analysis indicated that true yield values were approximated well by the number of electrons removed in non-oxygenation reactions. Experiments for estimating the kinetic parameters for utilization of the phenolic compounds were designed to eliminate the effects of the key cosubstrates of oxygenation reactions, O2, and the reduced intracellular electron carrier, NADH + H+. The maximum specific rate of substrate utilization, qmax, and the half-maximum rate concentration, K, for phenol and DCP were estimated. The kinetics for DCP were much slower than those for phenol, and the largest effect was a half-maximum rate concentration, which was 19 times larger for DCP. The larger K for DCP explains why DCP removal was low and sensitive to the solids retention time.

Anaerobic and aerobic biodegradation of chlorophenols using UASB and ASG bioreactors

Chlorophenol degradation was studied by combined anaerobic–aerobic treatments as a single or multi-substrate system. 2,4-Dichlorophenol (2,4-DCP) was degraded to the extent of 52 and 78% in up-flow anaerobic sludge blanket (UASB) and aerobic suspended growth (ASG) reactors respectively, at organic loading rates of 0.18 kg/m3/day and hydraulic retention time of 26.4 h in the presence of glucose. The UASB represents the dominating facultative anaerobic microbial population. When the effluent from the anaerobic reactor (UASB) was subjected to aerobic treatment on the ASG reactor, 2,4-DCP and COD removals of 86 and 95% respectively were achieved. Aerobic degradation of chlorophenol by acclimated mixed bacterial isolates was found to be sequential: 2-Chlorophenol (2-CP) and 4-CP were degraded first, followed by 2,4-DCP and 2,4,6-Trichlorophenol (2,4,6-TCP) while the contrary was obtained in anaerobic degradation. In anaerobic degradation by acclimated mixed bacterial cells, 2,4-DCP and 2,4,6-TCP were degraded first followed by mono-chlorophenols. The anaerobic/aerobic bioreactors were most efficient when operated in sequence (series) rather than in parallel.

Analysis of oxygenation reactions in a multi-substrate system—A new approach for estimating substrate-specific true yields

Analysis of oxygenation reactions in a multi-substrate system—A new approach for estimating substrate-specific true yields

A simple method to study the effectiveness of bioremediation aided, pump-and-treat technology for aquifers contaminated by non-aqueous phase liquids. II. Multi-component systems

Environmental contaminants are frequently encountered as mixtures of non-aqueous phase liquids (NAPLs). The dissolution of organic mixtures from the aquifer has been examined with and without biodegradation. Various physical processes involved have been quantified based on the assumptions that liquid-liquid and sorption equilibria are established at the beginning of each flushing; oxygen required for biochemical oxidation is completely consumed by the end of each flushing; and the rate of biochemical oxidation obeys the Monod kinetics for a multi-substrate system, characterized by an oxygen utilization factor. The mass fraction of any component remaining in the aquifer, its aqueous concentration, and the composition of the NAPL have been obtained as functions of the number of flushings (volume of the flushing solution/volume of the aqueous-phase voids). The results of the simulation with the model demonstrate that highly soluble components of the NAPL are mainly removed by the pump-and-treat mechanism while the components of extremely low solubility are unavailable to the microbes as substrates in a multi-component scenario. Bioremediation, however, transforms a significant proportion of the low solubility compounds after the more soluble components have been removed from the aquifer. The results also demonstrate that the rate of removal of compounds is retarded by the inclusion of a non-soluble component in the mixture.

Multisubstrate Micha�lis-Menten kinetics: Explicit dependence of substrate concentration on time for batch reactors

The problem of expressing the concentration of each of a set of substrates in a batch reactor as an explicit function of time in the case the multisubstrate system is described by Michaelis-Menten kinetics is tackled in dimensionless form via expansion as a Taylor series. The general analytical form for the coefficients of this expansion is obtained and estimates of the average quadratic error associated with increasing number of terms of the expansion is calculated. Considering the situation where the concentration of all substrates varies in a similar fashion, one concludes that use of four terms yields errors well within the range acceptable for predesign steps. In addition to other applications, the reasoning developed here finds applications in the statistical design and analysis of experiments aiming at the nonlinear fitting of kinetic parameters to actual data.

Measurement of choline acetyltransferase with [14C]acetate by a cycling procedure.

A multiple enzyme and multisubstrate cycling system is described for the radiometric determination of cholineacetyltransferase (ChAT) activity in crude tissue homogenates. The methods employs [14C]acetate coupled with the enzymes acetate kinase (AK) and phosphotransacetylase (PTA) for the generation of [14C]acetyl CoA. By recycling it was possible to avoid product inhibition of ChAT by CoA, ATP was maintained constant by rephosphorylation of ADP. Kinetics of the individual enzyme reactions were studied and the parameters obtained were used to select appropriate conditions to maintain linearity of varying amounts ChAT activity over a sixty minute time course. The sensitivity of the method is limited only by the specific activity of commercially available isotope labeled acetate.

The macrokinetic behavior of an enzymatic system with an enzyme inactivated in the reaction

This study deals with the behavior of a heterogeneous multisubstrate enzymatic system under enzyme inactivation in a reaction. Electronic computer modeling data have been obtained for its macrokinetics at different modes: (1) under mixed inflow of the substrates and (2) under their spatial separation. The enzymatic membrane exhibits low sensitivity to a change in the external conditions as the substrates are intermixed on the boundary. Quite the contrary, in the case of spatial separation of the substrates, the product flow from the membrane has displayed abrupt fluctuations at different boundary conditions. This work also looks into the arrangement of the reaction zones in the membrane and their transitions under different conditions.

6123. Computer-controlled four-gun multisubstrate sputtering system for the preparation of composition-modulated structures

6123. Computer-controlled four-gun multisubstrate sputtering system for the preparation of composition-modulated structures

The macrokinetics of an enzymatic system containing an enzyme that inactivates in the course of reaction

The work describes the action of a heterogenous multisubstrate enzymatic system under conditions involving inactivation of the enzyme. A nonsteady-state kinetic model has been suggested for description of the system. It has been found that the time dependence of the product flow from the membrane is a curve with a maximum which falls on a time equal to the reverse inactivation constant. It has been shown that the efficiency of such systems increases as does the time of the operation of the enzyme.

Influence of oxygen and substrate concentrations on the ideal film thickness and the maximum overall substrate uptake rate in microbial film fermenters

AbstractThe criterion for the oxygen limitation of substrate uptake in microbial film fermenters is expressed in terms of diffusion coefficients, utilization coefficients, and the free solution concentrations of substrate and oxygen. It is proposed that the ideal film thickness in such fermenters is equal to the penetration depth of the limiting substrate. The ideal film thickness is calculated, in terms of the parameters contained in the criterion for oxygen limitation, for three separate kinetic rate expressions. It is found that for the air–glucose–microbe system a simplified kinetic rate expression can be used and the region of dependence on two substrates is shown to be very limited. This is not true for other systems. Maximum uptake rates are calculated for a range of concentrations. Finally, it is shown that the procedure used can be generalized to determine the limiting substrate in a multisubstrate system and to calculate ideal film thickness and uptake rates for any pair of substrates where the kinetics of substrate uptake are known for the individual microorganism.

Simulation of Water Quality in Tarawera River

The Tarawera River, New Zealand, receives effluent from two paper and pulp mills and exhibits a very high rate of deoxygenation. Attempts have been made to predict water quality in the river using the Streeter-Phelps and various other first order models but none of these can successfully predict the observed concentrations of dissolved oxygen. Investigations of the biological activity in the river have indicated that biomass accumulates in the river sediments and that aerobic metabolism of these organisms on the organic matter in the overlying waters causes the high rate of deoxygenation. A stratified mathematical model is described which uses the Monod equations to predict the rates of growth of bacteria and protozoa in the river sediments. Chemical analyses of the mill effluent are used to formulate a theoretical multisubstrate system that predicts concentrations of dissolved oxygen successfully at varying rates of flow.