Automation of Biodegradation Tests

The feasibility of automating biodegradation tests was evaluated by comparing the results obtained using an Anachem SK233 automation system linked to an HPLC, and the traditional shake flask method using a shaker incubator and manual sampling. For the purpose of this study, several series of biodegradation kinetics were performed using a range of halogenated compounds which are common starting materials in organic synthesis. The first experiment involved two series of Fluorocinnamic Acid biodegradation kinetic tests performed over a period of 5 days. Several initial concentrations were used. Results obtained using the automated and manual methods were in very good agreement, with variations in absolute concentrations and kinetic constants ranging, between 10 % and 27 %. Half‐life values calculated from kinetic data ranged between 3.6 and 25.6 hours, depending on the initial organic compound concentrations (range 5 to 100 mg L^–1) and the activity of the activated sludge employed. In the second part of this study, the degradation of Bromobenzoic Acid, Chlorobenzoic Acid and Iodobenzoic Acid by activated sludge were investigated. Significant differences in the lag periods were observed. Differences in the hydrodynamics and the configuration of the vessels may contribute to the variations observed. However, it was found that for 4‐Iodobenzoic Acid, manual and automated methods yielded comparable first order kinetic values for the exponential growth bacterial cycle. 4‐Bromobenzoic Acid, 4‐Chlorobenzoic Acid and 4‐Iodobenzoic Acid rates of disappearance were estimated and complete removal on the shake flasks was observed after 72, 66 and 96 hours respectively. The results are very promising and open a lot of possibilities for future automation of a range of biodegradation and inhibition tests.     

Biodegradation of aromatic carboxylic acids by Pseudomonas mira

Abstract Biodegradation of aromatic acids (ferulic, vanillic and sipapinic acids) by the soil bacterium Pseudomonas mira was studied by high-pressure liquid chromatography. The presence of glucose in the culture medium slowed down the degradation process but did not affect its mechanism. In addition to vanillic acid and hydroquinone, the products of degradation were found to include acetophenone derivatives. Probably, a mechanism capable of shortening the side chain by spontaneous decarboxylation of unstable 3- keto -3-phenylpropionic acid was present, in addition to the elimination of acetic acid via degradation of the cinnamic acid-type compounds.     

Biodegradation kinetics of LAS in river water

This article reports the biodegradation kinetics of linear alkylbenzene sulfonates (LAS) in river water. The authors used the ‘river die-away' test method and high performance liquid chromatography to monitor LAS concentrations as functions of time in a series of tests systems. Controlled variables included initial LAS concentrations and incubation temperature. The kinetic parameters computed from the experimental data demonstrated strong correlations (r²>0.99) with theoretical values computed from the kinetic model presented in this paper. The proposed model accurately predicts concentrations of non-biodegradable substrate and the maximum specific microbial growth rate.     

Biodegradation kinetics of peptone and 2,6-dihydroxybenzoic acid by acclimated dual microbial culture

This study evaluated the kinetics of simultaneous biodegradation of peptone mixture and 2,6-dihydroxybenzoic acid (2,6-DHBA) by an acclimated dual microbial culture under aerobic conditions. A laboratory-scale sequencing batch reactor was sustained at steady-state with peptone mixture feeding. During the study period, peptone mixture feeding was continuously supplemented with 2,6-DHBA. Related experimental data were derived from three sets of parallel batch reactors, the first fed with the peptone mixture, the second with 2,6-DHBA and the third one with the two substrates, after acclimation of microbial culture and simultaneous biodegradation of both organics. A mechanistic model was developed for this purpose including the necessary model components and process kinetics for the model calibration of relevant experimental data. Model evaluation provided all biodegradation characteristics and kinetics for both peptone mixture and 2,6-DHBA. It also supported the development of a dual microbial community through acclimation, with the selective growth of a second group of microorganisms specifically capable of metabolizing 2,6-DHBA as an organic carbon source.     

Biodegradation of phthalic acid esters (PAEs) in soil bioaugmented with acclimated activated sludge

The degradation characteristics of four phthalic acid esters (PAEs), i.e. di-methyl phthalate (DMP), di-ethyl phthalate (DEP), di-n-butyl phthalate (DBP) and di-n-octyl phthalate (DOP) in the soil augmented with acclimated sludge was investigated in order to assess the efficacy of bioaugmentation as a strategy for remediating PAEs-contaminated soil and correlate the degradation rate of PAEs with their alkyl chain length. The results demonstrated that PAEs with shorter alkyl chain, that is, DMP and DEP could be degraded more quickly than DBP and DOP. The degradation of four PAEs in the soil conformed to a first-order reaction kinetic equation. The half-lives of PAEs degradation decreased significantly with increasing carbon number of the alcohol moiety. Half-lives decreased from 2.29 days for DMP to 28.4 days for DOP when the carbon number of alkyl chain increased from one for DMP to eight for DOP. The degradation rate of PAEs and the corresponding half-lives could correlate with the alkyl chain length and their octanol–water partition coefficients (K_ow) quite well for the four PAEs tested in this study.     

The effect of high magnetic field characterized by kinetics: Enhancing the biodegradation of acid red 1 with a strain of Bacillus sp.

The induced strain HG1 exposed to 10.0 T high magnetic field for 30 min and the original strain G1 (Bacillus sp.) isolated from soil were used to degrade acid red 1. The degradation process of the azo dye by these microbes was approximated by a reversed S-like curve. A kinetics equation whose parameters reflected the degradation process, was then established. The comparison and analysis of the kinetics parameters showed that the high magnetic field enhanced the biodegradation ability of the exposed strain—increasing the value of A (degradation constant) and V_max (maximum reaction rate) to 1.7 and 1.5 times, respectively, and at the same time, reducing the value of t_c (time when the maximum reaction rate was reached) to 0.8 times. Additionally, the UV/vis spectra also showed that the degradation ability of Bacillus sp. was improved and the metabolic pathway of the bacteria might be changed after being exposed to the high magnetic field.     

Biodegradation of 8-anilino-1-naphthalenesulfonic acid by Pseudomonas aeruginosa

Pseudomonas aeruginosa, isolated from soil near tannery effluent was able to degrade 8-anilino-1-naphthalenesulfonic acid (ANSA), a sulfonated aromatic amine. The organism degraded this amine up to a concentration of 1,200 mg l⁻¹ using glucose and ammonium nitrate as carbon and nitrogen sources respectively. The degradation started when the organism reached its late exponential growth phase. Salicylic acid and β-ketoadipic acid were identified as intermediate compounds using HPLC and GC–MS and provide evidence for ortho pathway reactions. Further proof for the pathway is obtained from the dioxygenase activity of the strain growing exponentially in medium with ANSA and glucose.     

Characterisation of bacterial cultures enriched on the chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid

The aim of this study was to enrich and characterise bacterial consortia from soils around a herbicide production plant through their capability to degrade the herbicides 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) and 4-(4-chloro-2-methylphenoxy) butyric acid (MCPB). Partial 16S rRNA gene sequencing revealed members of the genera Stenotrophomonas, Brevundimonas, Pseudomonas, and Ochrobactrum in the 2,4-DB- and MCPB-degrading communities. The degradation of 2,4-DB and MCPB was facilitated by the combined activities of the community members. Some of the members were able to utilise other herbicides from the family of chlorophenoxyalkanoic acids. During degradation of 2,4-DB and MCPB, phenol intermediates were detected, indicating ether cleavage of the side chain as the initial step responsible for the breakdown. This was also verified using an indicator medium. Repeated attempts to amplify putatively conserved tfd genes by PCR indicated the absence of tfd genes among the consortia members. First step cleavage of the chlorophenoxybutyric acid herbicides is by ether cleavage in bacteria and is encoded by divergent or different tfd gene types. The isolation of mixed cultures capable of degrading 2,4-DB and MCPB will aid future investigations to determine both the metabolic route for dissimilation and the fate of these herbicides in natural environments.     

A gas chromatography-mass spectrometry method for the measurement of fatty acid omega and omega(-1) hydroxylation kinetics by CYP4A1 using an artificial membrane system

A gas chromatography-mass spectrometry assay method for the analysis of lauric, myristic, and palmitic acids and their omega and omega(-1) hydroxylated metabolites from in vitro incubations of cytochrome P450 CYP4A1, involving solid-phase extraction and trimethysilyl derivatization, was developed. The assay was linear, precise, and accurate over the range 0.5 to 50microM for all the analytes. It has the advantages of a more rapid analysis time, an improved sensitivity, and a wider range of analytes compared with other methods. An artificial membrane system was optimized for application to purified CYP4A1 enzyme by investigating the molar ratios of cytochrome b(5) and cytochrome P450 reductase present in the incubation mixture. Using this method, the kinetics of omega and omega(-1) oxidation of lauric, myristic, and palmitic acids by CYP4A enzymes were measured and compared in rat liver microsomes and an artificial membrane system.     

Biodegradation of polyhydroxyalkanoates

Abstract Degradation of poly(3-hydroxybutyrate) and copolymers with 3-hydroxyvaleric acid was investigated in natural environments, and the microorganisms involved were isolated and identified. The influence of abiotic and biotic factors on the degradation is discussed.     

Synthesis of trans-4-Amino-1-hydroxy-2-methyl-2-butene from Isoprene

A novel synthetic pathway to trans-4-amino-1-hydroxy-2-methyl-2-butene (7), a useful synthetic intermediate of zeatin, is presented here. On selective monophthalimide formation, the trans-1, 4-dibromo-2-methyl-2-butene (10) prepared from isoprene (1) predominantly gives trans-1-bromo-2-methyl-4-phthalimido-2-butene (11). The compound (11) is converted to 7 via trans-1-acetoxy-2-methyl-4-phthalimido-2-butene (6). The overall yield of 7 from 1 is 33.6%. Base-catalyzed hydrolysis of 11 also gives 7 directly. Zeatin can be prepared by the condensation of 7 with 6-chloropurine.     

链霉菌HP-S-01降解高效氯氰菊酯的特性及其动力学

研究不同接菌量、温度、pH、装液量和农药初始浓度对链霉菌HP-S-01降解高效氯氰菊酯的影响。结果表明,在接菌量为0.6 g/L、28°C、pH 7.5和装液量为50 mL/250 mL三角瓶条件下培养3 d,该链霉菌对100 mg/L高效氯氰菊酯降解率达到96%以上。链霉菌HP-S-01还能明显降解高效氟氯氰菊酯、高效氯氟氰菊酯、右旋苯醚菊酯和胺菊酯等拟除虫菊酯农药,且降解过程符合一级动力学模型,降解半衰期分别为0.78、0.88、1.08和1.24 d。采用Andrews方程对链霉菌HP-S-01降解高效氯氰菊酯的过程进行拟合,其动力学参数为qmax=1.826 3 d?1,Ks=58.951 3 mg/L,Ki=359.378 2 mg/L,该链霉菌降解高效氯氰菊酯最佳的初始浓度为145.553 5 mg/L,试验数据与该动力学方程拟合较好。     

Biodegradation of diesel fuel by a microbial consortium in the presence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues

Fast development of ionic liquids as gaining more and more attention valuable chemicals will undoubtedly lead to environmental pollution. New formulations and application of ionic liquids may result in contamination in the presence of hydrophobic compounds, such as petroleum mixtures. We hypothesize that in the presence of diesel fuel low-water-soluble ionic liquids may become more toxic to hydrocarbon-degrading microorganisms. In this study the influence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues (side-chain length from C₃ to C₁₈) on biodegradation of diesel fuel by a bacterial consortium was investigated. Whereas test performed for the consortium cultivated on disodium succinate showed that toxicity of the investigated ionic liquids decreased with increase in side-chain length, only higher homologues (C₈–C₁₈) caused a decrease in diesel fuel biodegradation. As a result of exposure to toxic compounds also modification in cell surface hydrophobicity was observed (MATH). Disulphine blue active substances method was employed to determine partitioning index of ionic liquids between water and diesel fuel phase, which varied from 1.1 to 51% for C₃ and C₁₈ homologues, respectively. We conclude that in the presence of hydrocarbons acting as a solvent, the increased bioavailability of hydrophobic homologues is responsible for the decrease in biodegradation efficiency of diesel fuel.     

Biodegradation of 4-chlorobiphenyl by Micrococcus species

A Micrococcus sp., isolated by enrichment culture, grew on 4-chlorobiphenyl at 2 g/l as sole carbon source and produced 4-chlorobenzoic acid in the culture medium as a dead-end metabolite. The organism degraded 4-chlorobiphenyl by 2,3-dihydroxylation followed by meta-ring cleavage to yield 4-chlorobenzoate and carbon fragments for cell growth.     

红树林细菌Rhodococcus ruber1K降解邻苯二甲酸二丁酯的研究

A di-n-butyl phthalate (DBP)degrading bacterium Rhodococcus ruber was isolated from mangrove soil,and its degrading characteristics were studied.The results showed that the bacterium could grow well on the substrate with DBP as the sole source of carbon and energy,and the DBP of 50 mg稬^-1 could be completely degraded after 48 h.Under aerobic condition,the tentative pathway proposed for DBP degradation was through monoester initially,then phthalic acid,and finally CO₂ and H₂O.     

Dechlorination and spectral changes associated with bacterial degradation of 2-(2-methyl-4-chlorophenoxy)propionic acid

Summary The bacterial degradation of mecoprop (2-(2-methyl-4-chlorophenoxy)propionic acid) was studied using a mixed culture under aerobic conditions. The release of chlorine from mecoprop indicated incomplete degradation (75%), which did not proceed to completion upon extended incubation. The UV absorbance initially increased and this was associated with spectral distortion of the shoulder and trough regions and a slight shift in the maximum wavelength of absorption. GC-MS analysis indicated that 4-chloro-2-methylphenol was an intermediate in the degradative pathway of mecoprop. The GC-MS data also suggested the formation of other phenolic compounds with repositioned chloro-and methylgroups.     

Bile acid kinetics in man studied by radio thin-layer chromatography and densitometry coupling

A method based on coupling of the techniques of radioscanning a TLC plate and densitometry has been developed for the determination of pool sizes and fractional turnover rate of bile acids in man after intraduodenal administration of ¹⁴C-labelled acid. The validity of the method has been checked by comparison of the results obtained with those of an enzymatic spectrophotometric analysis, and a measurement of the radioactivity by liquid scintillation counting, after elution of the separated bile acid from a TLC plate. Advantages of the proposed method over the previous one include a reduced number of manipulations, the possibility of automation, a better reproducibility, and the possibility of elaborating the radiometric data obtained for the primary bile acid for better characterising its metabolism inside the enterohepatic circulation.     

Biodegradation of N-Methylpyrrolidone by Paracoccus sp. NMD-4 and its degradation pathway

N-Methylpyrrolidone (NMP), a kind of nitrogen-containing heterocyclic pollutant, is widely used in chemical industry. Microbial degradation is an important environmental fate process in soil and water, however, the microbial metabolic mechanism is still unknown. Strain NMD-4, capable of utilizing NMP as the sole source of carbon and nitrogen, was isolated from the activated sludge of a pesticide plant in Jiangsu, China, and identified as Paracoccus sp. based on its physiological–biochemical properties, as well as 16S rRNA gene sequence analysis. The degradation characteristic of NMP by strain NMD-4 was studied in a liquid culture, and the metabolic pathway of NMP by the strain was investigated. Two metabolites, 1-methyl-2,5-pyrrolidinedione and succinic acid, were detected and identified by liquid chromatography-mass spectrometry analysis, and a plausible microbial degradation pathway of NMP was proposed by the first time.     

Biodegradation of phenol and 4-chlorophenol by the yeast <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Biodegradation of phenol and 4-chlorophenol (4-cp) using a pure culture of Candida tropicalis was studied. The results showed that C. tropicalis could degrade 2,000 mg l⁻¹ phenol alone and 350 mg l⁻¹ 4-cp alone within 66 and 55 h, respectively. The capacity of the strain to degrade phenol was obviously higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Phenol beyond 800 mg l⁻¹ could not be degraded in the presence of 350 mg l⁻¹ 4-cp. Comparatively, low-concentration phenol from 100 to 600 mg l⁻¹ supplied a sole carbon and energy source for C. tropicalis in the initial phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in the fact that 4-cp biodegradation velocity was higher than that without phenol. And the capacity of C. tropicalis to degrade 4-cp was increased up to 420 mg l⁻¹ with the presence of 100–160 mg l⁻¹ phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and mixed substrates in batch cultures. The results illustrated that the models proposed adequately described the dynamic behaviors of biodegradation by C. tropicalis.