Modeling speciation effects on biodegradation in mixed metal/chelate systems

A new model, CCBATCH, comprehensively couples microbially catalyzed reactions to aqueous geochemistry. The effect of aqueous speciation on biodegradation reactions and the effect of biological reactions on the concentration of chemical species (e.g. H₂CO₃, NH ₄ ⁺ , O₂) are explicitly included in CCBATCH, allowing systematic investigation of kinetically controlled biological reactions. Bulk-phase chemical speciation reactions including acid/base and complexation are modeled as thermodynamically controlled, while biological reactions are modeled as kinetically controlled. A dual-Monod kinetic formulation for biological degradation reactions is coupled with stoichiometry for the degradation reaction to predict the rate of change of all biological and chemical species affected by the biological reactions. The capability of CCBATCH to capture pH and speciation effects on biological reactions is demonstrated by a series of modeling examples for the citrate/Fe(III) system. pH controls the concentration of potentially biologically available forms of citrate. When the percentage of the degradable substrate is low due to complexation or acid/base speciation, degradation rates may be slow despite high concentrations of substrate Complexation reactions that sequester substratein non-degradable forms may prevent degradation or stopdegradation reactions prior to complete substrate utilization. The capability of CCBATCH to couple aqueous speciation changes to biodegradation reaction kinetics and stoichiometry allows prediction of these key behaviors in mixed metal/chelate systems.     

Influence of physiological conditions on EDTA degradation

Aerobic biodegradation of the chelating agent EDTA by a mixed bacterial culture was investigated. Bacterial growth and degradation of the substrate required the presence of sufficient metal ions in the culture fluid. Uncomplexed EDTA interacted negatively with the cell walls of the bacteria and completely inhibited bacterial growth, whereas Mg(II)/Ca(II)-EDTA was degraded up to an initial concentration of 4.7 g/l. Therefore, concentrations of metal ions must be stoichiometric to that of EDTA or higher. Specific degradation rates ranged between 120 mg EDTA g^–1 (cell dry weight) h^–1 and 285 mg EDTA g^–1 h^–1. In contrast, complexes with high thermodynamic stability constants such as Fe(III)-EDTA remained as inert compounds in the solution. Specific growth rates of the mixed culture were found to vary between 0.03 h^–1 and 0.07 h^–1, which could be explained by population dynamics within the synergistic mixed community. Growth was significantly accelerated by the addition of vitamins.     

Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Co²⁺, Cd²⁺, Zn²⁺, Cu²⁺, or Fe³⁺. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10¹⁶ (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate,chelated ferric iron or nitrate

Flow-through aquifer columns were used to investigate the feasibility of adding sulfate, EDTA–Fe(III) or nitrate to enhance the biodegradation of BTEX and ethanol mixtures. The rapid biodegradation of ethanol near the inlet depleted the influent dissolved oxygen (8 mg l^-1), stimulated methanogenesis, and decreased BTEX biodegradation efficiencies from >99% in the absence of ethanol to an average of 32% for benzene, 49% for toluene, 77% for ethylbenzene, and about 30% for xylenes. The addition of sulfate, EDTA–Fe(III) or nitrate suppressed methanogenesis and significantly increased BTEX biodegradation efficiencies. Nevertheless, occasional clogging was experienced by the column augmented with EDTA–Fe(III) due to iron precipitation. Enhanced benzene biodegradation (>70% in all biostimulated columns) is noteworthy because benzene is often recalcitrant under anaerobic conditions. Influent dissolved oxygen apparently played a critical role because no significant benzene biotransformation was observed after oxygen was purged out of the influent media. The addition of anaerobic electron acceptors could enhance BTEX biodegradation not only by facilitating their anaerobic biodegradation but also by accelerating the mineralization of ethanol or other substrates that are labile under anaerobic conditions. This would alleviate the biochemical oxygen demand (BOD) and increase the likelihood that entraining oxygen would be used for the biotransformation of residual BTEX.     

Coordination isomers of biological iron transport compounds. III. (1) Transport of lambda-cis-chromic desferriferrichrome by Ustilago sphaerogena

Microbial iron transport studies of the structure and conformation dependent ferrichrome uptake system in Ustilago sphaerogena have been limited previously to kinetically labile metal ions such as the native ferrichrome complex and the aluminum(III) and gallium(III) analogs. Although two coordination isomers are possible (λ-cis amd δ-cis), no information can be obtained concerning their biological activity using kinetically labile complexes. In this report, both the ligand and chromic ion moieties of kinetically inert λ-cis-chromic [¹⁴C]-desferriferrichrome are shown to be taken up in Ustilago sphaerogena at rates comparable to that of ferrichrome. The λ-cis coordination isomer must be therefore at least one of the biologically active isomers and the transport system cannot rely on the rapid isomerization or dissociation of the labile ferric complex.     

Biological and chemical interaction of oxygen on the reduction of Fe(III)EDTA in a chemical absorption–biological reduction integrated NO x removal system

A promising chemical absorption–biological reduction integrated process has been proposed. A major problem of the process is oxidation of the active absorbent, ferrous ethylenediaminetetraacetate (Fe(II)EDTA), to the ferric species, leading to a significant decrease in NO removal efficiency. Thus the biological reduction of Fe(III)EDTA is vitally important for the continuous NO removal. Oxygen, an oxidizing agent and biological inhibitor, is typically present in the flue gas. It can significantly retard the application of the integrated process. This study investigated the influence mechanism of oxygen on the regeneration of Fe(II)EDTA in order to provide insight on how to eliminate or decrease the oxygen influence. The experimental results revealed that the dissolved oxygen and Fe(III)EDTA simultaneously served as electron acceptor for the microorganism. The Fe(III)EDTA reduction activity were directly inhibited by the dissolved oxygen. When the bioreactor was supplied with 3% and 8% oxygen in the gas phase, the concentration of initial dissolved oxygen in the liquid phase was 0.28 and 0.68 mg l⁻¹. Correspondingly, the instinct Fe(III)EDTA reduction activity of the microorganism determined under anoxic condition in a rotation shaker decreased from 1.09 to 0.84 and 0.49 mM h⁻¹. The oxidation of Fe(II)EDTA with dissolved oxygen prevented more dissolved oxygen access to the microorganism and eased the inhibition of dissolved oxygen on the microorganisms.     

Mathematical modeling of precipitation and dissolution reactions in microbiological systems

We expand the biogeochemical model CCBATCH to include a precipitation/dissolution sub-model that contains kinetic and equilibrium options. This advancement extends CCBATCH's usefulness to situations in which microbial reactions cause or are affected by formation or dissolution of a solid phase. The kinetic option employs a rate expression that explicitly includes the intrinsic kinetics for reaction ormass-transport control, the differencefrom thermodynamic equilibrium, and the aqueous concentration of the rate-limiting metal or ligand. The equilibrium feature can be used alone, and it also serves as check that the kinetic rate never is too fast and ``overshoots' equilibrium. The features of the expanded CCBATCH are illustrated by an example in which the precipitation of Fe(OH)_{3 (s)} allows the biodegradation of citric acid, even though complexes are strong and not bioavailable. Precipitation releases citrate ligand, and biodegradation of the citrate increases the pH.     

Arsenic precipitation in the bioleaching of enargite by Sulfolobus BC at 70 °C

Enargite (Cu₃AsS₄) was leached at 70°C by Sulfolobus BC in shake-flasks. The highest copper dissolution (52% after 550 h of leaching) was obtained with bacteria and 1 g l^–1 ferric ion. In the absence of ferric ion, Sulfolobus BC catalyzes the bioleaching of enargite through a direct mechanism after adhesion onto the mineral surface. In ferric bioleaching, arsenic precipitated as ferric arsenate and arsenic remained associated to the solid residues, preventing the presence of a high dissolved arsenic concentration in the leaching solution. About 90% inhibition of bacterial growth rate and activity was observed for dissolved arsenic concentrations above 600 mg l^–1 for As(III) and above 1000 mg l^–1 for As(V). Arsenic-bearing copper ores and concentrates could be leached by Sulfolobus BC in the presence of ferric iron due to the favourable precipitation of arsenic ion as ferric arsenate, avoiding significant bacterial inhibition.     

Role of apoplast acidification by the H+ pump

We have studied the mechanism of the response to iron deficiency in rape (Brassica napus L.), taking into account our previous results: net H⁺ extrusion maintains a pH shift between the root apoplast and the solution, and the magnitude of the pH shift decreases as the buffering power in the solution increases. The ferric stress increased the ability of roots to reduce Fe[III]EDTA. Buffering the bulk solution (without change in pH) inhibited Fe[III]EDTA reduction. At constant bulk pH, the inhibition (ratio of the Fe[III]EDTA-reduction rates measured in the presence and in the absence of buffer) increased with the rate of H⁺ extrusion (modulated by the length of a pretreatment in 0.2 mM CaSO₄). These results support the hypothesis that the apoplastic pH shift caused by H⁺ excretion stimulated Fe[III] reduction. The shape of the curves describing the pH-dependency of Fe[III]EDTA reduction in the presence and in the absence of a buffer fitted this hypothesis. When compared to the titration curves of Fe[III]citrate and of Fe[III]EDTA, the curves describing the dependency of the reduction rate of these chelates on pH indicated that the stimulation of Fe[III] reduction by the apoplastic pH shift due to H⁺ excretion could result from changes in electrostatic interactions between the chelates and the fixed chargers of the cell wall and-or plasmalemma. Blocking H⁺ excretion by vanadate resulted in complete inhibiton of Fe[III] reduction, even in an acidic medium in which there was neither a pH shift nor an inhibitory effect of a buffer. This indicates that the apoplastic pH shift resulting from H⁺ pumping is not the only mechanism which is involved in the coupling of Fe[III] reduction to H⁺ transport. Our results shed light on the way by which the strong buffering effect of HCO ₃ ^- in some soils may be involved in iron deficiency encountered by some of the plants which grow in them.     

Evidence for Microbial Fe(III) Reduction in Anoxic, Mining-Impacted Lake Sediments (Lake Coeur d'Alene, Idaho)

Mining-impacted sediments of Lake Coeur d'Alene, Idaho, contain more than 10% metals on a dry weight basis, approximately 80% of which is iron. Since iron (hydr)oxides adsorb toxic, ore-associated elements, such as arsenic, iron (hydr)oxide reduction may in part control the mobility and bioavailability of these elements. Geochemical and microbiological data were collected to examine the ecological role of dissimilatory Fe(III)-reducing bacteria in this habitat. The concentration of mild-acid-extractable Fe(II) increased with sediment depth up to 50 g kg⁻¹, suggesting that iron reduction has occurred recently. The maximum concentrations of dissolved Fe(II) in interstitial water (41 mg liter⁻¹) occurred 10 to 15 cm beneath the sediment-water interface, suggesting that sulfidogenesis may not be the predominant terminal electron-accepting process in this environment and that dissolved Fe(II) arises from biological reductive dissolution of iron (hydr)oxides. The concentration of sedimentary magnetite (Fe₃O₄), a common product of bacterial Fe(III) hydroxide reduction, was as much as 15.5 g kg⁻¹. Most-probable-number enrichment cultures revealed that the mean density of Fe(III)-reducing bacteria was 8.3 × 10⁵ cells g (dry weight) of sediment⁻¹. Two new strains of dissimilatory Fe(III)-reducing bacteria were isolated from surface sediments. Collectively, the results of this study support the hypothesis that dissimilatory reduction of iron has been and continues to be an important biogeochemical process in the environment examined.     

EDTA activation of H2 photoproduction by Rhodospirillum rubrum

Summary The effect of trace elements (Fe, Ni) and chelating compounds on the activity of hydrogen (H₂) uptake (Hup) hydrogenase, nitrogenase and rate and yield of H₂ photoproduction from l-lactate in photosynthetic cultures of Rhodospirillum rubrum was investigated. Hup activity depended on the availability of Ni²⁺ and was inhibited by EDTA (0.3–0.5 mm ethylenedinitrilotetraacetic acid). Addition of EDTA (0.5 mm) to the culture medium caused a nearly complete inactivation of Hup activity and activation of nitrogenase, which was paralleled by a threefold increase in total H₂ photoproduced from lactate. Hup^– mutants, isolated by transposon Tn5 mutagenesis, produced maximally twofold more H₂ than the wild-type. Experiments with different chelating agents [EDTA, NTA (nitrilotriacetic acid), citrate, isocitrate] and varying concentrations of Fe²⁺ and Fe³⁺ showed that photosynthetic growth and nitrogenase activity of R. rubrum were strongly influenced by the iron supply. It is concluded that EDTA enhanced H₂ photoproduction by (I) inhibition of biosynthesis of Hup hydrogenase and (II) mobilization of iron, thereby activating the biosynthesis of the nitrogenase complex. Correspondence to: M. Kern     

Uptake and degradation of EDTA by Escherichia coli

It was found that Escherichia coli exhibited a growth by utilization of Fe(III)EDTA as a sole nitrogen source. No significant growth was detected when Fe(III)EDTA was replaced by EDTA complexes with other metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. When EDTA uptake was measured in the presence of various ions, it was remarkable only when Fe³⁺ was present. The cell extract of E. coli exhibited a significant degradation of EDTA only in the presence of Fe³⁺. It is likely that the capability of E. coli for the growth by utilization of Fe(III)EDTA results from the Fe³⁺-dependent uptake and degradation of EDTA.     

The solution of manganese oxide by iron sources used in nutrient solutions

Summary The solubility of two manganese oxides was measured in 5×10^–5 to 15×10^–5 M iron and organic acid solutions. The oxides were soluble in all the 15 × 10^–5 M solutions tested except ferric chloride. The amount of manganese dissolved by mixtures of the iron and acid solutions was greater than the sum of that dissolved by the separate solutions. It was suggested that ferric chloride should be used as the iron source in critical studies of the availability of manganese oxides in sand cultures.     

The influence of ferrous-complexed EDTA as a solubilization agent and its auto-regeneration on the removal of nitric oxide gas through the culture of green alga Scenedesmus sp

The influence of iron-complexed ehylenediaminetetraacetic acid (EDTA) was studied on nitric oxide (NO) removal using photoautotropic cultivation of green alga Scenedesmus. Fe(II)EDTA is an active solubilization agent of NO in water, while the oxidized Fe(III)EDTA is not. When a gas mixture containing 300 ppm NO was treated through the Scenedesmus culture containing 5 mM Fe(II)EDTA, a constant level of 80–85% NO removal was achieved for a prolonged period. A certain fraction of Fe(II)EDTA remained without being oxidized to Fe(III)EDTA because of the existence of reversible oxidation–reduction balance between Fe(II)EDTA and Fe(III)EDTA. When Fe(III)EDTA was added to the culture instead of Fe(II)EDTA, Fe(II) was generated via reduction of Fe(III), resulting in the increase of NO removal and cell density. This was possible because of the generated Fe(II)EDTA which contributed to the dissolution of NO. Therefore, a long-term NO removal was possible with Fe(III)EDTA, as well as with Fe(II)EDTA, in the present microalgal system. The supplementation of free EDTA was necessary to extend the period of NO removal because EDTA is consumed by biodegradation while the decrease of total iron content was not significant.     

Theoretical investigation of the ring opening process of verdoheme to biliverdin in the presence of dioxygen

The conversion of ferrous verdoheme to ferric biliverdin in the presence of O₂ was investigated using the B3LYP method. Both 6-31G and 6-31G (d) basis sets were employed for geometry optimization calculation as well as energy stabilization estimation. Three possible pathways for the conversion of iron verdoheme to iron biliverdin were considered. In the first route oxygen and reducing electron were employed. In this path formation of ferrous verdoheme-O₂ complex was followed by the addition of one electron to the ferrous-oxycomplex to produce ferric peroxide intermediate. The ferric peroxide intermediate experienced an intramolecular nucleophilic attack to the most positive position at 5-oxo carbons on the ring to form a closed ring biliverdin. Subsequently the ring opening process took place and the iron (III) biliverdin complex was formed. Closed ring iron biliverdin intermediate and open ring iron biliverdin formed as a product of verdoheme cleavage were respectively 13.20 and 32.70 kcal mol⁻¹ more stable than ferric peroxide intermediate. Barrier energy for conversion of ferric peroxide to closed ring Fe (III) biliverdin and from the latter to Fe (III) biliverdin were respectively 8.67 and 3.35 kcal mol⁻¹. In this path spin ground states are doublet except for iron (III) biliverdin in which spin state is quartet. In the second path a ferrous-O₂ complex was formed and, without going to a one electron reduction process, nucleophilic attack of iron superoxide complex took place followed by the formation of iron (III) biliverdin. This path is thermodynamically and kinetically less favorable than the first one. In addition, iron hydro peroxy complex or direct attack of O₂ to macrocycle to form an isoporphyrin type intermediate have shown energy surfaces less favorable than aforementioned routes.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Redox Cycling of Iron Supports Growth and Magnetite Synthesis by Aquaspirillum magnetotacticum

Under anaerobic conditions and in the absence of alternative electron acceptors, growth of the magnetic bacterium Aquaspirillum magnetotacticum MSI was iron concentration dependent. Weak chelation of the iron (with quinate, oxalate, or 2,3-dihydroxybenzoate) enhanced growth, whereas strong chelation (with EDTA, citrate, or nitrilotriacetic acid) retarded the growth of strain MSI relative to that of controls lacking chelators. Growth was proportional to the percentage of unchelated iron in medium containing EDTA in various molar ratios to iron. Addition of the respiratory inhibitors antimycin A (5 μM), NaCN (10 mM), and NaN₃ (10 mM) inhibited growth with Fe(III) or NO₃^- as the terminal electron acceptor. Growth with O₂ and NO₃^- was inhibited by 2-heptyl-4-hydroxyquinolone-N-oxide (HOQNO) but not with 2 mM Fe(III). Under strongly reducing conditions, strain MS1 survived but grew poorly and became irreversibly nonmagnetic. Growth and iron reduction in anaerobic cultures were stimulated by the provision of small amounts of O₂ or H₂O₂. Slow infusion of air to cultures which had reduced virtually all of the Fe(III) in the medium (2 mM) supported a high rate of iron reoxidation (relative to killed controls) and growth in proportion to the amount of iron reoxidized. Oxygen consumption by iron-reducing cultures was predominantly biological, since NaCN and HOQNO both inhibited consumption. Inhibition of oxygen consumption (and iron reoxidation) by the addition of ferrozine and the inhibition of iron oxidation (and oxygen consumption) by the addition of HOQNO suggest that iron oxidation by strain MS1 is an aerobic respiratory process, perhaps tied to energy conservation. Iron oxidation was also necessary for magnetite synthesis, since in microaerobic denitrifying cultures, sequestration of reduced iron by ferrozine present in 10-fold molar excess to the available iron resulted in loss of magnetism and a severe drop in the average magnetosome number of the cells.     

Influence of Complex Structure on the Biodegradation of Iron-Citrate Complexes

The biodegradation of iron-citrate complexes depends on the structure of the complex formed between the metal and citric acid. Ferric iron formed a bidentate complex with citric acid, [Fe(III) (OH)₂ cit]^2- involving two carboxylic acid groups, and was degraded at the rate of 86 μM h^-1. In contrast, ferrous iron formed a tridentate complex with citric acid, [Fe(II) cit]^-, involving two carboxylic acid groups and the hydroxyl group, and was resistant to biodegradation. However, oxidation and hydrolysis of the ferrous iron resulted in the formation of a tridentate ferric-citrate complex, [Fe(III)OH cit]^-, which was further hydrolyzed to a bidentate complex, [Fe(III)(OH)₂ cit]^2-, that was readily degraded. The rate of degradation of the ferrous-citrate complex depended on the rate of its conversion to the more hydrolyzed form of the ferric-citrate complex. Bacteria accelerated the conversion much more than did chemical oxidation and hydrolysis.     

Spatial variability in the naphthalene mineralization response to oxygen, nitrate, and orthophosphate amendments in MGP aquifer sediments

The feasibility of aerobic in situ bioremediation isbeing investigated for use in a strategy to controlsubsurface coal tar contamination at the site of aformer manufactured gas plant. As part of thisinvestigation, anoxic aquifer sands collected between11 and 25 m below ground surface were assayed in batchmicrocosms to measure the singular and combinedeffects of O₂, NO₃ ^-, andPO₄ ^3- on ¹⁴C-naphthalenemineralization. The influence of these additivesvaried considerably between sediments. A high initialconcentration of O₂ (21 mg/L) promoted thegreatest extent of mineralization in the majority ofactive sediments. NO₃ ^- (85 mg/L) wasobserved to enhance, inhibit, or have no effect on therate of naphthalene mineralization, althoughsignificant denitrification was observed in nearly allthe active sediments. Data suggest thatPO₄ ^3- complexation and/or precipitation withsediment cations limited P bioavailability. Thesediments that were incapable of mineralizingnaphthalene were characterized by low pH (< 4.1),high SO₄ ^2- (> 500 mg/L), and moderate tohigh dissolved Fe(II) (30–265 mg/L) whenequilibrated aerobically with water. Fe(II) likelyexerted a significant O₂ demand that reduced theO₂ available as an electron acceptor forbiodegradation. These experiments demonstrate thatwhile aeration/oxygenation can be an effectivestrategy for enhancing subsurface bioremediation ofaromatic hydrocarbons, the biodegradation response toaeration/oxygenation and nutrient addition may varyconsiderably within an aquifer.     

An NADH: Fe(III) EDTA oxidoreductase from Cryptococcus albidus: an enzyme involved in ethylene production in vivo?

An ethylene-forming enzyme which forms ethylene from 2-oxo-4-methylthiobutyric acid (KMBA) was purified to an electrophoretically homogeneous state from a cell-free extract of Cryptococcus albidus IFP 0939. The presence of KMBA, NADH, Fe(III) chelated to EDTA and oxygen were essential for the formation of ethylene. When ferric ions, as Fe(III)EDTA, in the reaction mixture were replaced by Fe(II)EDTA under aerobic conditions, the non-enzymatic formation of ethylene was observed. Under anaerobic conditions in the presence of Fe(III)EDTA and NADH, the enzyme reduced 2 mol of Fe(III) with 1 mol of NADH to give 2 mol of Fe(II) and 1 mol NAD+, indicating that the ethylene-forming enzyme is an NADH-Fe(III)EDTA oxidoreductase. The role of NADH:Fe(III)EDTA oxidoreductase activity in the formation in vivo ethylene from KMBA is discussed.