Slow complexation kinetics for ferric iron and EDTA complexes make EDTA non-biodegradable

Published experimental data on ethylenediaminetetraacetic acid (EDTA) biodegradation in the presence of ferric iron (Fe(III)) showed that rapid biodegradation of EDTA suddenly stopped, leaving a residual of unbiodegraded EDTA that was equal to the concentration of dissolved Fe(III). We hypothesize that slow kinetics for the dissociation of two iron-EDTA complexes - FeEDTA^- and FeOHEDTA^2- – sequestered the EDTA in a form that is biologically unavailable. To evaluate this hypothesis, we added to the biogeochemical model CCBATCH a new sub-model for kinetically controlled complexation. CCBATCH simulations with kinetically controlled complexation for FeEDTA^- and FeOHEDTA^2- and the observed concentration of total dissolved Fe(III) accurately predicted the sudden cessation of EDTA biodegradation at the exact time shown experimentally. Our simulations also correctly predicted the observed residual EDTA concentration and the amounts of biomass and NH₄ ⁺. Alternate explanations for the experimental results – strong equilibrium complexation of ferric iron and EDTA and precipitation of calcium and magnesium solids – could not capture the observed trends. This analysis using CCBATCH's new sub-model for kinetically controlled complexation shows that EDTA, once it becomes complexed with Fe(III), becomes biologically unavailable.     

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.     

A biogeochemical framework for metal detoxification in sulfidic systems

We develop a comprehensive biogeochemical framework for understanding and quantitatively evaluating metals bio-protection in sulfidic microbial systems. We implement the biogeochemical framework in CCBATCH by expanding its chemical equilibrium and biological sub-models for surface complexation and the formation of soluble and solid products, respectively. We apply the expanded CCBATCH to understand the relative importance of the various key ligands of sulfidic systems in Zn detoxification. Our biogeochemical analysis emphasizes the relative importance of sulfide over other microbial products in Zn detoxification, because the sulfide yield is an order of magnitude higher than that of other microbial products, while its reactivity toward metals also is highest. In particular, metal-titration simulations using the expanded CCBATCH in a batch mode illustrate how sulfide detoxifies Zn, controlling its speciation as long as total sulfide is greater than added Zn. Only in the absence of sulfide does complexation of Zn to biogenic organic ligands play a role in detoxification. Our biogeochemical analysis conveys fundamental insight on the potential of the key ligands of sulfidic systems to effect Zn detoxification. Sulfide stands out for its reactivity and prevalence in sulfidic systems.     

Production profiles of phenolics from fungal tannic acid biodegradation in submerged and solid-state fermentation

Potent antioxidant phenolics are derived from tannin biodegradation. Understanding of biodegradation pathways through the identification of the intermediates molecules of great value like tannins is important to pursuit the production of bioactive monomers. Biodegradation of tannins remains poorly understood due to their chemical complexity and reactivity. Tannic acid biodegradation by Aspergillus niger GH1 in submerged fermentation (SF) and solid state fermentation (SSF) was evaluated by liquid chromatography coupled to mass spectrometry (LC–MS). Both cultures were kinetically monitored for the biodegradation profiles during 72 h. Differences in tannic acid composition were evidenced and the consumption of substrate and identification of biodegradation intermediates were achieved. The mechanism of tannic acid degradation by A. niger GH1 is by degradation of high molecular weight gallotannins and highly polymerized tannins to small molecules like gallic acid, digalloyl glucose and trigalloyl glucose. Important differences on time of substrate uptake and product release were revealed.     

Coupling hydrocarbon degradation to anaerobic respiration and mineral diagenesis: theoretical constraints

The diagenetic mineral assemblages in petroleum reservoirs control the formation fluid pH and pCO₂. Anaerobic biodegradation of petroleum is controlled by the transfer of electrons from reduced organic species to inorganic, redox sensitive, aqueous and mineral species in many cases through intermediates such as H₂ and CH₃COO^?. The terminal electron accepting reactions induce the dissolution or precipitation of the same minerals that control the ambient pH and pCO₂ in petroleum reservoirs. In this study, we develop a model for anaerobic biodegradation of petroleum that couples the production of acetate and H₂ to ‘late stage’ diagenetic reactions. The model reveals that the principal terminal electron accepting process and electron donor control the type of diagenetic reaction, and that the petroleum biodegradation rate is controlled through thermodynamic restriction by the minimum ΔG required to support a specific microbial metabolism, the fluid flux and the mineral assemblage. These relationships are illustrated by modeling coupled microbial diagenesis and biodegradation of the Gullfaks oil reservoir. The results indicate that the complete dissolution of albite by acids generated during oil biodegradation and the corresponding elevated pCO₂ seen in the Gullfaks field are best explained by methanogenic respiration coupled to hydrocarbon degradation and that the biodegradation rate is likely controlled by the pCH₄. Biodegradation of Gullfaks oil by a consortium that includes either Fe³⁺‐reducing or ‐reducing bacteria cannot explain the observed diagenetic mineral assemblage or pCO₂. For octane, biodegradation, not water washing, was the principal agent for removal at fluid velocities <20 m Myr^?1.     

The inhibitory effect of cyclodextrin on the degradation of 9α-hydroxyandrost-4-ene-3,17-dione by Mycobacterium sp. VKM Ac-1817D

The inhibitory effect of methylated β-cyclodextrin (mCD) on steroid degradation was studied using the degradation of 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) by Mycobacterium sp. VKM Ac-1817D as a model process. The formation of the [9-OH-AD–mCD] complex was shown by ¹H NMR-spectroscopy. The biodegradation of 9-OH-AD by whole and disrupted cells was carried out at 30°C in aqueous solutions with or without mCD. Enzyme kinetic parameters were calculated by non-linear regression of the Michaelis–Menten plot. The complexation of 9-OH-AD and mCD was evaluated via the stability constant for the [9-OH-AD–mCD] complex. The V_max and K_M values calculated for the free (non-complex) steroid in mCD solutions corresponded to steroid degradation in the absence of mCD. The inclusion complex [9-OH-AD–mCD] was shown to be resistant to enzymatic degradation. The inference is made that the ‘‘guest–host’’ molecular complexation with cyclodextrin can be used for the control of steroid bioconversions.     

Synthesis, isolation, spectroscopic and structural characterization of a new pH complex structural variant from the aqueous vanadium(V)-peroxo-citrate ternary system

In a pH-specific fashion, V₂O₅, citric acid and H₂O₂ reacted at pH 5.5-6.0 and afforded a red crystalline product at 4 °C. Elemental analysis pointed to the molecular formulation . Complex 1 was further characterized by UV/Vis, FT-IR, NMR, cyclic voltammetry, and X-ray crystallography. The X-ray structure of 1 reveals two dinuclear vanadium-peroxo-citrate subunits, A and B, linked through a hydrogen bond. In both A and B, the citrate ligands have different protonation states, ultimately affording a pentagonal bipyramidal geometry around each V(V) ion. The peroxide ligands bind V(V) in a side-on fashion. pH-Dependent, non-thermal and thermal transformations of 1 unravel its connection with key participants in the vanadium-peroxo-citrate ternary system and project its association with other non-peroxo binary complexes of variable vanadium oxidation state, geometry, citrate binding mode and state of protonation. Overall, the surprising twist in the aqueous synthetic chemistry of the investigated ternary system: (a) projects a new pH structural variant (species A) as a component of the speciation; (b) provides an in-depth look at that speciation under specific pH conditions; and (c) offers significant insight into the aqueous structural speciation of vanadium with peroxide and citrate, and its potential relevance to biological processes.     

Effect of Concentration of Organic Chemicals on Their Biodegradation by Natural Microbial Communities

The effect of concentration on the biodegradation of synthetic organic chemicals by natural microbial communities was investigated by adding individual ¹⁴C-labeled organic compounds to stream water at various initial concentrations and measuring the formation of ¹⁴CO₂. The rate of degradation of p-chlorobenzoate and chloroacetate at initial concentrations of 47 pg/ml to 47 μg/ml fell markedly with lower initial concentrations, although half or more of the compound was converted to CO₂ in 8 days or less. On the other hand, little mineralization of 2,4-dichlorophenoxyacetate and 1-naphthyl-N-methylcarbamate, or the naphthol formed from the latter, occurred when these compounds were present at initial concentrations of 2 to 3 ng/ml or less, although 60% or more of the chemical initially present at higher concentrations was converted to CO₂ in 6 days. It is concluded that laboratory tests of biodegradation involving chemical concentrations greater than those in nature may not correctly assess the rate of biodegradation in natural ecosystems and that low substrate concentration may be important in limiting biodegradation in natural waters.     

Benzo[a]pyrene degradation using simultaneously combined chemical oxidation,biotreatment with Fusarium solani and cyclodextrins

The interest of simultaneously combining chemical (Fenton’s reaction) and biological treatments for the degradation of a high molecular weight polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) has been studied in laboratory tests. An optimal concentration of 1.5 × 10⁻³ M H₂O₂ as Fenton’s reagent was firstly determined as being compatible with the growth of Fusarium solani, the Deuteromycete fungus used in the biodegradation process. For the enhancement of BaP solubilisation, cyclodextrins were also used in the performed tests. The best degradation performance was achieved through the use of 5 × 10⁻³ M hydroxypropyl-β-cyclodextrin (HPBCD) in comparison with randomly methylated-β-cyclodextrin (RAMEB). When Fenton’s treatment was combined with biodegradation, a beneficial effect on BaP degradation (25%) was obtained in comparison with biodegradation alone (8%) or with chemical oxidation alone (16%) in the presence of HPBCD for 12 days of incubation.     

Transport issues and bioremediation modeling for the in situ aerobic co-metabolism of chlorinated solvents

For aerobic co-metabolism of chlorinated solvents to occur, it isnecessary that oxygen, a primary substrate, and the chlorinated compound all be available to an appropriate microorganism – that is, a microorganism capable of producing the nonspecific enzyme that will promote degradation of the ontaminant while the primary substrate is aerobically metabolized. Thus, the transport processes that serve to mix the reactants are crucial in determining the rate and extent of biodegradation, particularly when considering in situ biodegradation. These transport processes intersect, at a range of scales, with the biochemical reactions. This paper reviews how the important processes contributing to aerobic co-metabolism of chlorinated solvents at different scales can be integrated into mathematical models. The application of these models to field-scale bioremediation is critically examined. It is demonstrated that modeling can be a useful tool in gaining insight into the physical, chemical, and biological processes relevant to aerobic co-metabolism, designing aerobic co-metabolic bioremediation systems, and predicting system performance. Research needs are identified that primarily relate to gaps in our current knowledge of inter-scale interactions.     

Enhancement of biodegradability of disposable polyethylene in controlled biological soil

Plastics as polyethylene are widely used in packaging and other agricultural applications. They accumulate in the environment at a rate of 25 million tons per year. Thus, the development and use of degradable plastics was proposed as a solution for plastic waste problem. Because of the ever-increasing use of plastic films, nowadays, biodegradability has become a useful characteristic for plastics. Conversely, the introduction of biodegradable plastics has generated a need for methods to evaluate the biodegradation of these polymers in landfills and solid waste treatment systems such as composting or anaerobic digestion treatment plants. The purpose of this study was to investigate the biodegradation of disposable low-density polyethylene bags containing starch (12%), autoxidizable fatty acid ester and catalytic agents in soil. Structurally this work intended to evaluate the capacity of Phanerochaete chrysosporium (ATCC 34541) to enhance polyethylene film biodegradation in soil microcosms. Soil samples inoculated with P. chrysosporium were mixed with LDPE/starch blend films and biological changes of the films and soil were monitored for 6 months. The biodegradation of polyethylene starch blend film has been determined by the physical, chemical and biological properties of the samples such as pH, biomass, CO₂ formation, percentage elongation, relative viscosity and FTIR spectrum.     

Bio-pretreatment of municipal solid waste prior to landfilling and its kinetics

The conventional landfilling does not promote sustainable waste management due to uncontrolled emissions which potentially degrade the environment. Pretreatment of municipal solid waste prior to landfilling significantly enhances waste stabilization, reduces the emissions and provides many advantages. Therefore, pretreatment of municipal solid waste methods were investigated. The major objectives of biological pretreatment are to degrade most easily degradable organic matters of MSW in a short duration under controlled conditions so as to produce desired quality for landfill. To investigate the suitable pretreatment method prior to landfilling for developing countries four pretreatment simulators were developed in the laboratory: (i) anaerobic simulator (R₁), (ii) aerobic pretreatment simulator by natural convection of air (R₂), (iii) aerobic pretreatment simulator by natural convection of air with leachate recirculation (R₃) and (iv) forced aeration and leachate recirculation (R₄). During the pretreatment organic matter, elemental composition, i.e., carbon, hydrogen, nitrogen and settlement were determined for bench scale experiments. A two-component kinetic model is proposed for the biodegradation of organic matter. Biodegradation kinetic constants were determined for readily and slowly degradable organic matter. The biodegradation of organic matter efficiency in terms of kinetic rate constants for the pretreatment simulators was observed as R₄ > R₃ > R₂ > R₁. Biodegradation rate constants for readily degradable matter in simulators R₄ and R₃ were 0.225 and 0.222 per day. R₃ and R₄ simulators were more effective in reducing methane emissions about 45% and 55%, respectively, as compared to anaerobic simulator R₁. Pretreatment of MSW, by natural convection of air with leachate recirculation R₃ is sustainable method to reduce the emissions and to stabilize the waste prior to landfilling.     

Microbial degradation of tetraethyl lead in soil monitored by microcalorimetry

Sieved agricultural soil samples were treated with the anti-knock agent tetraethyl lead (Et₄Pb), and the resulting effects were analyzed by microcalorimetry. Et₄Pb additions resulted in an increase of the heat production rate, provided that oxygen was present and that the soil was not autoclaved. The increased heat production rate was accompanied by degradation of Et₄Pb, as verified by speciation analysis (GC-MS) of the remaining Et₄Pb and its ionic degradation products (triethyl lead and diethyl lead cations). Conclusive evidence was obtained that these transformations were mediated mainly by microbes. At an initial Et₄Pb concentration of 2 g Pb/kg dry weight the biodegradation rate was about 780 μmol day⁻¹ kg dry weight⁻¹, whilst the chemical decomposition was only 50 μmol day⁻¹ kg dry weight⁻¹. A fivefold rise of the initial Et₄Pb concentration resulted in a decrease of the biodegradation rate to 600 μmol day⁻¹ kg dry weight⁻¹ and an increase of the chemical decomposition to 200 μmol day⁻¹ kg dry weight⁻¹. The biodegradation rate was not influenced by the addition of glucose, which means that no indication for a cometabolic attack of Et₄Pb was found. Received: 25 February 1997 / Received revision: 22 April 1997 / Accepted: 27 April 1997     

Kinetically controlled acylation of 6-APA catalyzed by penicillin acylase from Streptomyces lavendulae: effect of reaction conditions in the enzymatic synthesis of penicillin V

Abstract

Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction conditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of 6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concentrations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled synthesis (KCS) of β-lactam antibiotics: penV concentration reached a maximum (highest yield or Y_max) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final penV concentration was abruptly reduced when basic pH values were employed in the reaction. Under the selected conditions (100?mM Tris/HCl buffer pH 7.0, 30?°C, 2.7% (v/v) DMSO, 20?mM MPOA, 0.3 UI/ml of SlPVA), Y_max was enhanced by increasing the substrate molar ratio (6-APA to MPOA) up to 5, reaching a maximum of 94.5% and a S/H value of 16.4 (ratio of synthetic activity to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has allowed us to obtain some of the highest Y_max and S/H values among those reported in literature for KCS of β-lactam antibiotics. Although many penicillin G acylases (PGAs) have been described in kinetically controlled acylations, SlPVA should be considered as a different enzyme in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different substrate specificity compared to PGAs.     

The application of papain,ficin and clostripain in kinetically controlled peptide synthesis in frozen aqueous solutions

The capability of the cysteine proteases ficin, papain and clostripain to form peptide bonds in frozen aqueous solutions was investigated. Freezing the reaction mixture resulted in increased peptide yields in kinetically controlled coupling of Bz–Arg–OEt with various amino acid amides and dipeptides. Under these conditions, peptide yields increased up to 70% depending on the enzyme and the amino component used. Enzyme-catalysed peptide syntheses were carried out under optimized reaction conditions (temperature, amino component concentration and pH before freezing) using the condensation of Bz–Arg–OEt and H–Leu–NH₂ as a model reaction.     

Mycoremediation of Endosulfan and Its Metabolites in Aqueous Medium and Soil by Botryosphaeria laricina JAS6 and Aspergillus tamarii JAS9

Microbial degradation offers an efficient and ecofriendly approach to remove toxicants from the contaminated environments. Botryosphaeria laricina JAS6 and Aspergillus tamarii JAS9 were capable of degrading endosulfan and their metabolites which were isolated through enrichment technique. Both the strains were able to withstand an exposure of 1300 mg/L and showed luxuriant growth at 1000 mg/L of endosulfan. The change in pH in the culture broth was from 6.8 to 3.4 and 3.8 during growth kinetic studies of JAS6 and JAS9 strains, respectively upon biological degradation of endosulfan. The degradation of endosulfan by JAS6 and JAS9 strains were examined by HPLC. The biodegradation rate constant (k) and the initial concentration were reduced by 50% (DT₅₀) which was determined by first and pseudo first order kinetic models. In the present investigation it has been revealed that Botryosphaeria laricina JAS6 and Aspergillus tamarii JAS9 possessing endosulfan degrading capability are being reported for the first time. These findings confirm the degradation of endosulfan by JAS6 and JAS9 strains which were accompanied by significant reduction in the toxicity and could be used as remedial measure in contaminated environments.     

Kinetics of phenol degradation using Pseudomonas putida MTCC 1194

Pseudomonas putida (MTCC 1194) has been used to degrade phenol in water in the concentration range 100–1000?ppm. The inhibition effects of phenol as substrate have become predominant above the concentration of 500?ppm (5.31?mmoles/dm³). The optimum temperature and initial pH required for maximum phenol biodegradation were 30?°C and 7.00 respectively. From the degradation data the activation energy (E _a ) was found to be equal to 13.8?kcal/g mole substrate reacted. The most suitable inoculum age and volume for highest phenol degradation were 12?hrs and 7% v/v respectively. Surfactants had negligible effect on phenol biodegradation process for this microorganism. Monod model has been used to interpret the free cell data on phenol biodegradation. The kinetic parameters have been estimated upto initial concentration of 5.31?mmoles/dm³. μ _max and K _S gradually increased with higher concentration of phenol. However, beyond the phenol concentration of 5.31?mmoles/dm³, the inhibition became prominant. The μ _max has been to be a strong function of initial phenol concentration. The simulated and the experimental phenol degradation profiles have good correspondence with each other.     

Relationships of respiratory quotient to microbial biomass and hydrocarbon contaminant degradation during soil bioremediation

This work focused on monitoring respiratory quotient, RQ (defined as a ratio of CO₂ production to O₂ uptake rates), microbial growth and residual hydrocarbon concentration during bioremediation experiments performed on laboratory soil microcosms. The aim of the study was to determine if the time course biodegradation profile of the contaminant can be related to the RQ evolution and to investigate the effect of the water content on RQ measurements. A natural soil was artificially contaminated with hexadecane and adjusted with inorganic nutrients to stimulate biodegradation. Microbial growth, CO₂ production, O₂ uptake and residual hexadecane were periodically monitored at different soil water contents ranging from 0.15 to 0.35 g water g⁻¹ of dry soil. Results showed that microbial activity and contaminant degradation were strongly dependent on soil water content. Maximal growth and hexadecane depletion were obtained at a water content of 0.20 g water g⁻¹ of dry soil, which corresponded to 46.6% of the water holding capacity. Hexadecane degradation was considerably reduced with increasing soil water content. RQ values fluctuated as a function of the hexadecane biodegradation phases. The lowest RQs corresponded to the highest hexadecane depletion and microbial growth. The water content variation did not significantly affect the shape of the RQ evolution curves as a function of time. It only modified the magnitude of RQ values. This study indicates that additional biological and chemical analyses are needed to support RQ data when monitoring contaminant degradation to have an accurate understanding of all the biotic processes, which may occur simultaneously.     

Fe(II)/α-Ketoglutarate-Dependent Hydroxylases and Related Enzymes

Fe(II)/α-ketoglutarate (αKG)-dependent hydroxylases catalyze an amazing diversity of reactions that result in protein side-chain modifications, repair of alkylated DNA/RNA, biosynthesis of antibiotics and plant products, metabolism related to lipids, and biodegradation of a variety of compounds. These enzymes possess a β-strand “jellyroll” structural fold that contains three metal-binding ligands found in a His¹-X-Asp/Glu-X_n-His² motif. The cosubstrate, αKG, chelates Fe(II) using its C-2 keto group (binding opposite the Asp/Glu residue) and C-1 carboxylate (coordinating opposite either His¹ or His²). Oxidative decomposition of αKG forms CO₂ plus succinate and leads to the generation of an Fe(IV)-oxo or other activated oxygen species that hydroxylate the primary substrate. The reactive oxygen species displays alternate reactivity in related enzymes that catalyze desaturations, ring expansions, or ring closures. Other enzymes resemble the Fe(II)/αKG-dependent hydroxylases in terms of protein structure or chemical mechanism but do not utilize αKG as a substrate. This review describes the reactions catalyzed by this superfamily of enzymes, highlights key active site features revealed by structural studies, and summarizes results from spectroscopic and other approaches that provide insights into the chemical mechanisms.