Development and Evaluation of an Online CO2 Evolution Test and a Multicomponent Biodegradation Test System

Well-established biodegradation tests use biogenously evolved carbon dioxide (CO₂) as an analytical parameter to determine the ultimate biodegradability of substances. A newly developed analytical technique based on the continuous online measurement of conductivity showed its suitability over other techniques. It could be demonstrated that the method met all criteria of established biodegradation tests, gave continuous biodegradation curves, and was more reliable than other tests. In parallel experiments, only small variations in the biodegradation pattern occurred. When comparing the new online CO₂ method with existing CO₂ evolution tests, growth rates and lag periods were similar and only the final degree of biodegradation of aniline was slightly lower. A further test development was the unification and parallel measurement of all three important summary parameters for biodegradation—i.e., CO₂ evolution, determination of the biochemical oxygen demand (BOD), and removal of dissolved organic carbon (DOC)—in a multicomponent biodegradation test system (MCBTS). The practicability of this test method was demonstrated with aniline. This test system had advantages for poorly water-soluble and highly volatile compounds and allowed the determination of the carbon fraction integrated into biomass (heterotrophic yield). The integrated online measurements of CO₂ and BOD systems produced continuous degradation curves, which better met the stringent criteria of ready biodegradability (60% biodegradation in a 10-day window). Furthermore the data could be used to calculate maximal growth rates for the modeling of biodegradation processes.     

Biodegradation of phthalic acid esters in river water and activated sludge.

The primary and ultimate biodegradability of phthalic acid, monobutyl phthalate, and five structurally diverse phthalic acid ester plasticizers in river water and activated sludge samples were determined via ultraviolet spectrophotometry, gas chromatography, and CO2 evolution. The compounds studied underwent rapid primary biodegradation in both unacclimated river water and acclimated activated sludge. When activated sludge acclimated to phthalic acid esters was used as the inoculum for the CO2 evolution procedure, greater than 85% of the total theoretical CO2 was evolved. These studies demonstrate that the phthalic acid ester plasticizers and intermediate degradation products readily undergo ultimate degradation in different mixed microbial systems at concentrations ranging from 1 to 83 mg/liter.     

Priming effect as determined by adding 14C-glucose to modified controlled composting test

The development of new biodegradable packaging materials, especially biodegradable plastics, has created a need for biodegradability testing. The European standard for controlled composting test was used in this study for assessing if the addition of a test material results in excess CO₂ production in compost. This effect, designated as the priming effect, would give an erroneous result for biodegradation, which is based on CO₂ formation from the test material. Glucose was selected as a test substrate because it is the degradation product of starch and cellulose, which are major compounds of many packaging materials. Both ¹⁴C-glucose and non-labelled glucose was applied to nine compost samples of variable stability and agefrom two weeks to 1.5 years. CO₂ and ¹⁴CO₂ evolution were measured during the incubation. Biodegradation of glucose in unstable composts (age leq6 months) was negative and ¹⁴CO₂ evolution was poor, although the respective composts without glucose produced relatively high amounts of CO₂. It was concluded that a negative priming effect was observed in unstable composts, in which glucose remained mostly non-degraded and apparently inhibited the mineralization of native organic matter in the compost. In stable composts (age 6 months), biodegradation of glucose was high and approximately equal to ¹⁴C-glucose mineralization, i.e., the composts showed no priming effect. Young composts were unsuitable for controlled composting test due to lack of stability. It is important to ensure that the compost inoculum used for the test is sufficiently stable.     

BIODEGRADATION OF PHTHALATE ESTERS BY CYANOBACTERIA1

Phthalate esters (PEs) are endocrine‐disrupting pollutants that are ubiquitous in the environment and can be degraded by microorganisms. In this study, we investigated the kinetics and pathway of biodegradation of di‐n‐butyl phthalate (DBP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) by cyanobacteria Anabaena flos‐aquae G. S. West (strain 4054) and two strains of Microcystis aeruginosa (Kütz.) Kütz. (strain 2396 and strain SM). Gas chromatography/mass spectroscopy (GC/MS) and a deuterium‐labeled compound were used to analyze the degrading intermediates. The findings revealed that all three organisms were capable of metabolizing PE, and that among these organisms, A. flos‐aquae achieved the highest degradation. Additionally, the biodegradation of DBP, DEP, and DMP followed first‐order kinetics. Moreover, the results of the enzymatic study suggested that PE was degraded through transesterification on the side chains rather than deesterification. Finally, experiments using deuterium‐labeled DBP showed that there were two degradation pathways: C₁₆→ C₁₄→ C₁₂→ C₁₀→ C₈ and C₁₆→ C₁₅→ C₁₃→ C₁₁→ C₉. Based on our results, the biodegradation pathway of PE for cyanobacteria was suggested.     

Determination of aerobic biodegradation kinetics of olive oil mill wastewater

Aerobic biological treatment was conducted for the treatment of high strength olive oil mill wastewater (OMW). Two different approaches were used for kinetic modeling of OMW biodegradation. TOC removal and CO₂–C evolution were monitored in an open and a closed bioreactor systems, respectively. Gompertz, Refractory organics plus first-order (RFO) and Chen–Hashimoto equations were applied to estimate the kinetic parameters by using the data from bioreactors. Furthermore, change in oxidation stage of carbon was monitored and temperature dependency of OMW biodegradation was investigated based on activation energy. At room temperature, 64% of TOC was removed in the open bioreactor while cumulative CO₂–C evolution was 6.32 g L⁻¹ in closed the bioreactor. Higher biodegradation efficiency and kinetic parameters were obtained at 25 °C rather than 10 °C. Gompertz and RFO equations provided better fitting with CO₂–C and TOC data, respectively. Experimental and kinetic estimations indicated that OMW constituted of approximately 30% refractory organics. The comparison of two different modeling approaches showed that kinetic modeling based on CO₂–C provided better correlation with the experimental data. Temperature coefficient indicated that biological degradation of OMW is slightly dependent on temperature.     

Fungal biodegradation of dibutyl phthalate and toxicity of its breakdown products on the basis of fungal and bacterial growth

Phthalates are esters of phthalic acid that give flexibility to polyvinyl chloride. Diverse studies have reported that these compounds might be carcinogenic, mutagenic and/or teratogenic. Radial growth rate, biomass, hyphal thickness of Neurospora sitophyla, Trichoderma harzianum and Aspergillus niger, grown in two different concentrations of dibutyl phthalate (DBP) (500 and 1,000 mg/l) in agar and in submerged fermentation were studied. The inhibitory concentration (IC₅₀) and the constant of biodegradation of dibutyl phthalate in Escherichia coli cultures were used to evaluate toxicity. The radial growth rate and thickness of the hypha were positively correlated with the concentration of phthalate. The pH of the cultures decreased as the fermentation proceeded. It is shown that these fungi are able to degrade DBP to non-toxic compounds and that these can be used as sole carbon and energy sources by this bacterium. It is demonstrated that the biodegradation of the DBP is directly correlated with the IC₅₀. This is the first study that reports a method to determine the biodegradation of DBP on the basis of the IC₅₀ and fungal growth, and the effect of this phthalate on the growth and thickness of hyphae of filamentous fungi in agar and in submerged fermentation.     

Co-Metabolic Biodegradation of DBP by Paenibacillus sp. S-3 and H-2

Two di-n-butyl phthalate (DBP)-degrading strains, designated as S-3 and H-2, were isolated from DBP-polluted soil and both identified as Paenibacillus sp. When DBP was provided as the sole carbon source, about 45.5 and 71.7 % of DBP (100 mg/L) were degraded by strain S-3 and H-2, respectively, after incubation for 48 h. However, DBP (100 mg/L) was degraded completely by co-culture of strain S-3 and H-2 after incubation for 60 h. Four phthalic acid (PA) esters could be utilized by co-metabolism in the study and the degradation rates followed the order of dimethyl phthalate > diethyl phthalate > DBP > dioctyl phthalate. The metabolic pathway of DBP was elucidated based on the results of metabolites identification and enzyme assays. For strain S-3, DBP was degraded into butyl hydrogen phthalate which was degraded to PA by carboxyesterase further. But PA could be not hydrolyzed further because strain S-3 lacked 3,4-phthalate dioxygenase. Different with S-3, strain H-2 could hydrolyze PA into 3,4-dihydroxy-PA by 3,4-phthalate dioxygenase. Then 3,4-dihydroxy-PA was converted to protocatechuate and benzoic acid. Finally, the aromatic ring was cleavage and mineralized to CO₂ and H₂O. Above all, co-metabolism could increase the activity of 3,4-phthalate dioxygenase and accelerated the degradation of DBP. This study highlights an important potential use of co-metabolic biodegradation for the in situ bioremediation of DBP and its metabolites-contaminated environment.     

Activated Sludge Biodegradation of 12 Commercial Phthalate Esters

The activated sludge biodegradability of 12 commercial phthalate esters was evaluated in two test systems: (i) a semicontinuous activated sludge test and (ii) an acclimated 19-day die-away procedure. Both procedures demonstrated that phthalate esters are rapidly biodegraded under activated sludge conditions when loss of the parent phthalate ester (primary degradation) is measured.     

Biodegradation of diethyl phthalate by an organic-solvent-tolerant Bacillus subtilis strain 3C3 and effect of phthalate ester coexistence

The contamination and distribution of phthalate esters - synthetic compounds widely used in plastic product production, including food and medical packaging - has raised safety concerns due to their endocrine-disrupting activity and mandated to be treated. Bacillus subtilis strain 3C3, isolated as an organic-solvent-tolerant bacterium, was capable of utilizing diethyl phthalate as a sole carbon source. Biodegradation of diethyl phthalate occurred constitutively without lag period, and its kinetics followed a first-order model. The biodegradability was significantly enhanced with the supplementation of yeast extract as a co-metabolic substrate. In the presence of Tween-80 as a solubilizing agent, cells rapidly degrade a range of short-chain phthalate esters at high concentrations (up to 1000 mg l⁻¹ for diethyl phthalate). The biodegradation of short-chain phthalates in the binary, ternary and quaternary substrate system revealed that the coexistence of other short-chain phthalates had no significant influence on the biodegradation of diethyl phthalate, and vice versa. These results substantiated that B. subtilis strain 3C3 has potential application as a bioaugmented bacterial culture for bioremediation of phthalates.     

Biodegradation of nonionic and anionic surfactants in domestic wastewater under simulated sewer conditions

The ultimate disposition of chemicals discarded down the drain can be substantially impacted by their fate in the sewer, but to date limited data have been published on the biodegradability of chemicals in sewer systems. The recently established OECD 314 guideline (Simulation tests to assess the biodegradability of chemicals discharged in wastewater, 2008) contains a simulation method (314A) for evaluating the biodegradation of chemicals in sewage under simulated sewer conditions. This research used the OECD 314A method to evaluate the rates and pathways of primary and ultimate biodegradation of a suite of ¹⁴C-labeled homologues representing four classes of high volume surfactants including nonionic alkyl ethoxylates (AE), and anionic alkyl ethoxysulfates (AES), alkyl sulfate (AS) and linear alkyl benzene sulfonate (LAS). All the tested homologues exhibited >97 % loss of parent, formation of metabolites, and some level (16–94 %) of CO₂ production after being incubated 96–100 h in raw domestic wastewater. Comparison of C₁₂E₃, C₁₄E₃, and C₁₆E₃ showed that the first order biodegradation rate was affected by alkyl chain length with rates ranging from 6.8 h^?1 for C₁₂E₃ to 0.49 h^?1 for C₁₆E₃. Conversely, comparison of C₁₄E₁, C₁₄E₃, and C₁₄E₉ showed that the number of ethoxy units did not impact the biodegradation rate. AES and AS degraded quickly with first order kinetic rates of 1.9–3.7 and 41 h^?1 respectively. LAS did not exhibit first order decay kinetics and primary degradation was slow. Biodegradation pathways were also determined. This work shows that biodegradation in the sewer has a substantial impact on levels of surfactants and surfactant metabolites that ultimately reach wastewater treatment plants.     

Biodegradation of hydrocarbon cuts used for diesel oil formulation

The biodegradability of various types of diesel oil (DO), such as straight-run DO, light-cycle DO, hydrocracking DO, Fischer–Tropsch DO and commercial DO, was investigated in biodegradation tests performed in closed-batch systems using two microflorae. The first microflora was an activated sludge from an urban wastewater treatment plant as commonly used in biodegradability tests of commercial products and the second was a microflora from a hydrocarbon-polluted soil with possible specific capacities for hydrocarbon degradation. Kinetics of CO₂ production and extent of DO biodegradation were obtained by chromatographic procedures. Under optimised conditions, the polluted-soil microflora was found to extensively degrade all the DO types tested, the degradation efficiencies being higher than 88%. For all the DOs tested, the biodegradation capacities of the soil microflora were significantly higher than those of the activated sludge. Using both microflora, the extent of biodegradation was highly dependent upon the type of DO used, especially its hydrocarbon composition. Linear alkanes were completely degraded in each test, whereas identifiable branched alkanes such as farnesane, pristane or phytane were degraded to variable extents. Among the aromatics, substituted mono-aromatics were also variably biodegraded.     

Biodegradation of 2-sulfonato fatty acid methyl ester (α-SFMe): Identification of microorganisms isolated from activated sludge and their capability for degrading α-SFMe

Three bacterial strains, A, B and C, were isolated from activated sludge as 2-sulfonato-fatty-acid-methyl-ester (-SFMe)-degrading microorganisms. From the results of morphological, physiological and biochemical studies, and analyses of 16S rRNA gene sequences, isolate A was identified as Agrobacterium tumefaciens while B and C were Pseudomonas putida, respectively. To demonstrate their capability for the ultimate biodegradation of -SFMe, the degradation kinetics have been investigated using C₁₄--SFMe and 2-¹⁴C-labeled C₁₆--SFMe. The biodegradation was determined by measuring dissolved organic carbon (DOC) and released SO₄ ^2–, in the shake-culture test, and evolved ¹⁴CO₂ in the modified Organisation for Economic Co-operation and Development (OECD) test. In the shake culture test with C₁₄--SFMe, DOC removal was progressive throughout the test. Liberation of inorganic sulfate started after DOC removal and then rapidly increased. During the ¹⁴CO₂ evolution tests, the mineralization of radiolabeled carbon started quickly and reached about 80% of the initially added radioactivity at the end of the tests. The results obtained indicated that all of the isolates had the capability for ultimately degrading -SFMe through the oxidation of the alkyl carbons and desulfonation (cleavage of the C-S linkage).     

Fate of Hydrocarbons During Oily Sludge Disposal in Soil

A 1,280-day laboratory simulation of the “landfarming” process explored the fate in soil of polynuclear aromatics (PNAs) and total extractable hydrocarbon residues originating from the disposal of an oily sludge. In addition to the measurement of CO₂ evolution, periodic analyses of PNAs and hydrocarbons monitored biodegradation activity. The estimation of carbon balance and of soil organic matter assessed the fate of residual hydrocarbons. Seven sludge applications during a 920-day active disposal period were followed by a 360-day inactive “closure” period with no further sludge applications. A burst of CO₂ evolution followed each sludge addition, but substantial amounts of undegraded hydrocarbons remained at the end of the study. Hydrocarbon accumulation did not inhibit biodegradation performance. Conversion of hydrocarbons to CO₂ predominated during active disposal; incorporation into soil organic matter predominated during the closure period. In this sludge, the predominant PNAs were degraded more completely (85%) than total hydrocarbons. Both biodegradation and abiotic losses of three- and four-ring PNAs contributed to this result. Some PNAs with five and six rings were more persistent, but these constituted only a small portion of the PNAs in the sludge. The study confirmed that the microbially mediated processes of mineralization and humification remove sludge hydrocarbons from soils of landfarms with reasonable efficiency.     

Survey of the Anaerobic Biodegradation Potential of Organic Chemicals in Digesting Sludge

The degradation potential of 77 organic chemicals under methanogenic conditions was examined with an anaerobic digesting sludge from the United Kingdom. Degradation was assessed in terms of net total gas (CH₄ plus CO₂) produced, expressed as a percentage of the theoretical production (ThGP). The compounds tested were selected from various chemical groups and included substituted phenols and benzoates, pesticides, phthalic acid esters, homocyclic and heterocyclic ring compounds, glycols, and monosubstituted benzenes. The results obtained were in good agreement with published surveys of biodegradability in U.S. digesting sludges and other methanogenic environments. In general, the presence of chloro or nitro groups inhibited anaerobic gas production, while carboxyl and hydroxyl groups facilitated biodegradation. The relationship between substituent position and susceptibility to methanogenic degradation was compound dependent. The following chemicals were completely degraded (≥80% ThGP) at a concentration of 50 mg of carbon per liter: phenol, 2-aminophenol, 4-cresol, catechol, sodium benzoate, 4-aminobenzoic acid, 3-chlorobenzoic acid, phthalic acid, ethylene glycol, diethylene glycol, triethylene glycol, sodium stearate, and quinoline. 3-Cresol, 4-chlorobenzoic acid, dimethyl phthalate, and pyridine were partially degraded. Although the remaining chemicals tested were either persistent or toxic, their behavior may differ at more environmentally realistic chemical-to-biomass ratios. Our findings suggest that biodegradability assessments made with sludge from one source can be extrapolated to sludge from another source with a reasonable degree of confidence and should help in predicting the fate of an organic chemical during the anaerobic digestion of sewage sludge.     

Characterization of hydrocarbon-degrading bacteria isolated from oil-contaminated sediments in the Sultanate of Oman and evaluation of bioaugmentation and biostimulation approaches in microcosm experiments

Two oil-polluted sediments (PD and KH) were sampled from a coastal region in Oman for the isolation of hydrocarbon-degrading bacteria and for testing different bioremediation approaches. Fourty strains were isolated, eighteen were affiliated to Marinobacter whereas the rest belonged to Pseudomonas, Halomonas, Hahella and Alcanivorax. All strains grew well at 2–7% salinity and between 20 and 60 °C. The strains exhibited a better growth on long chain than on short chain alkanes. Biostimulation and bioaugmentation were compared in both sediments and oil biodegradation was followed by measuring CO₂ evolution and by gas chromatography (GC). The evolved CO₂ reached 0.45 ± 0.02 and 2.23 ± 0.07 mg CO₂ g⁻¹ sediment after 88 days in the untreated PD and KH sediments, respectively. While the addition of inorganic nutrients resulted in 1.2–3.7 fold increase in CO₂ evolution in both sediments, the addition of the bacterial consortium was only effective in the PD sediment. The maximum CO₂ evolution was measured when both nutrients and bacteria were added and this corresponded to a total oil mineralization of 2.6 ± 0.12 and 1.49 ± 0.04% of the initial oil after 88 days in the PD and KH sediments, respectively. GC analysis confirmed the CO₂ data and showed that most of the degraded compounds belonged to alkanes. We conclude that the Omani polluted sediments contain halotolerant and thermotolerant bacteria and biostimulation is more efficient than bioaugmentation for their cleanup.     

Effects of CO<Subscript>2</Subscript> on the formation of flavour volatiles during fermentation with immobilised brewer’s yeast

Immobilised-cell fermentors offer great benefits compared to traditional free-cell systems. However, a major problem is unbalanced flavour production when these fermentors are used for the production of alcoholic beverages. One of the keys to obtaining better control over flavour formation may be the concentration of dissolved CO₂, which has inhibitory effects on yeast growth and metabolism. This article demonstrates that the presence of immobilisation matrices facilitates the removal of CO₂ from the liquid medium, which results in a low level of dissolved CO₂ during fermentation. Moreover, the formation of volatile higher alcohols and esters was greatly enhanced in the immobilised-cell system when compared to the free cell system. By sparging a CO₂ flow (45 ml/min) into the immobilised-cell system, cell growth was reduced by 10–30% during the active fermentation stage, while the fermentation rate was unaffected. The uptake of branched-chain amino acids was reduced by 8–22%, and the formation of higher alcohols and esters was reduced on average by 15% and 18%, respectively. The results of this study suggest that mismatched flavour profiles with immobilised-cell systems can be adjusted by controlling the level of dissolved CO₂ during fermentation with immobilised yeast.     

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.     

Primary biodegradation of a series of alkyl sulfosuccinates by mixed bacterial culture

A mixed bacterial culture capable of primary biodegradation of sodium alkyl sulfosuccinates R¹-OOC−CH(SO₃Na)−CH₂−COO−R² was obtained from soil microorganisms by enrichment cultivation and adaptation in the presence of mono-n-dodecyl sulfosuccinate. Gram-negative psychrophilic bacteria with proteolytic, lipolytic and ammonifying activities were prevalent in the culture. The process of primary biodegradation of alkyl sulfosuccinates can be described by firstorder reaction kinetics. The rate constants for linear esters were ascending in the order C₄<C₅<C₆ (45 μmol/min per g cell protein) and further descending with increasing length of the carbon chain C₆>C₈>=C₁₃. Substitution of cyclohexyl for n-hexyl group resulted in fourfold decrease in biodegradation rate. Terminal branching of alkyl chain does not affect the rate of primary biodegradation.     

Properties of a Newly Identified Esterase from Bacillus sp. K91 and Its Novel Function in Diisobutyl Phthalate Degradation

The widely used plasticizer phthalate esters (PAEs) have become a public concern because of their effects on environmental contamination and toxicity on mammals. However, the biodegradation of PAEs, especially diisobutyl phthalate (DiBP), remains poorly understood. In particular, genes involved in the hydrolysis of these compounds were not conclusively identified. In this study, the CarEW gene, which encodes an enzyme that is capable of hydrolyzing ρ-nitrophenyl esters of fatty acids, was cloned from a thermophilic bacterium Bacillus sp. K91 and heterologously expressed in Escherichia coli BL21 using the pEASY-E2 expression system. The enzyme showed a monomeric structure with a molecular mass of approximately 53.76 kDa and pI of 4.88. The enzyme exhibited maximal activity at pH 7.5 and 45°C, with ρ-NP butyrate as the best substrate. The enzyme was fairly stable within the pH range from 7.0 to 8.5. High-pressure liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) were employed to detect the catabolic pathway of DiBP. Two intermediate products were identified, and a potential biodegradation pathway was proposed. Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.