Intrinsic Biodegradation Potential Research Articles

1,2-DCA biodegradation potential of an aquifer assessed in situ and in aerobic and anaerobic microcosms

Background1,2-dichloroethane (1,2-DCA) biodegradation can occur through aerobic or anaerobic pathways that can be exploited in bioremediation strategies. Bioremediation interventions are site specific and generally based on anaerobic pathways, nevertheless expanding knowledge on proper conditions favoring the biodegradation and especially on 1,2-DCA degrading microorganisms is crucial. In this work the intrinsic biodegradation potential of an aquifer impacted by Chlorinated Aliphatic Hydrocarbons (mainly 1,2-DCA) was evaluated by characterizing the aquifer microbiome across space and time and by setting up biostimulation treatments in microcosms under different aerobic and anaerobic conditions, in parallel.ResultsThe microbial profiling of the aquifer revealed noticeable alpha and beta diversity across the sampling sites within the aquifer and strong fluctuations over time. Surprisingly both the anaerobic and aerobic biostimulation treatments led to the successful removal of 1,2-DCA in microcosms, the enrichment of known 1,2-DCA degraders and the detection of reductive or hydrolytic dehalogenases. Ancylobacter and Starkeya were enriched in aerobic microcosms. Desulfovibrio and Desulfuromonas, known as perchloroethylene degraders, were enriched in anaerobic microcosms, suggesting they could be yet unknown 1,2-DCA respirers.ConclusionsOur results demonstrate the occurrence of both aerobic and anaerobic bioremediation potential in the aquifer despite its negative redox potential. Due to the feasibility of direct oxidation with oxygen insufflation, we propose that an enhanced bioremediation strategy based on direct oxidation of 1,2-DCA could be applied to the contaminated aquifer as an ecofriendly, efficient and cost-effective approach as an alternative to anaerobic biodegradation.

Ecotoxicity and bioremediation potential assessment of soil from oil refinery station area

PurposeThe aim of the present study was to evaluate the toxicity and biodegradation potential of oil hydrocarbons contaminated soil samples obtained from different depths at an oil refinery station area. An approach involving chemical, microbiological, respirometry and ecotoxicity assessment of soil polluted by oil hydrocarbons was adopted, in order to determine the biodegradability of pollutants and ecotoxicological effects of natural attenuation strategy.MethodsThe ecotoxicity of soil samples was evaluated using an ostracod test kit and a seed germination test. The results of the phytotoxicity assay were expressed as a percentage of seedling emergence and as the relative yield of fresh and dry biomass compared to control plants. The intrinsic biodegradation potential of the contaminated soil was examined using a Micro-Oxymax respirometer. Intrinsic biodegradation rates were estimated from the slopes of linear regressions curves plotted for cumulative O2 uptake. The obtained values were then entered in the mass balance equation for the stoichiometric reaction of hydrocarbon decomposition and converted per kg of soil per day.ResultsAlthough the tested contaminants were biodegradable in the respirometric assay, they were slightly to moderately toxic to plants and extremely toxic to ostracods. The noxious effects raised with the increased concentration of contaminants. The monocotyledonous oat was more tolerant to higher concentrations of oil hydrocarbons than the other test plants, indicating its greater suitability for soil reclamation purposes.ConclusionBy assessing phytotoxicity and effect on ostracod mortality and progress of soil self-decontamination process, proper approach of reclamation of demoted area can be provided.

Investigating the impact of inoculum source on anaerobic digestion of various species of marine macroalgae

This study investigated the intrinsic biodegradation potential of marine organic sediment for effective biogas production from various species of marine macroalgae and non-marine biomass. Biogas production potential tests were carried out on three species of seaweed harvested from the west coasts of Scotland, Laminaria digitata, Fucus serratus, and Saccharina latissima, and on a non-marine cellulose biomass seeded with uncultivated and unadapted anoxic marine sediments. As a comparison, the same experiments were repeated using the same substrates but seeded with active mesophilic anaerobically digested sewage sludge. For the cultures seeded with anoxic marine sediments, the highest methane yield was observed in both L. digitata and S. latissima cultures while F. serratus and cellulose, cultures performed relatively poorly. For those seeded with digested sludge, all cultures performed relatively well, except F. serratus. These results show that marine sediments can be effective inoculum for seaweeds digestion. Phylogenetic analyses of the methanogenic community in both sources of inoculum showed that the methanogen community within the sediment and sludge seeded cultures were different. Each culture was dominated by methanogenic populations suitable for the utilisation of the specific biomass derivatives and environmental conditions. For instance, members of the genus Methanosaeta which, dominated sludge seeded cultures were not detected in the sediment seeded cultures. A similar occurrence was observed for the genus Methanofollis which was only detected in the sediment seeded cultures. Hence, in areas where seaweed forms part of a co-digestion with non-marine biomass, start-up using a mixture of anoxic marine sediments and digested wastewater sludge has the potential to provide greater process stability and robustness than using either as sole inoculum.

Integrative isotopic and molecular approach for the diagnosis and implementation of an efficient in-situ enhanced biological reductive dechlorination of chlorinated ethenes

Based on the previously observed intrinsic bioremediation potential of a site originally contaminated with perchloroethene (PCE), field-derived lactate-amended microcosms were performed to test different lactate isomers and concentrations, and find clearer isotopic and molecular parameters proving the feasibility of an in-situ enhanced reductive dechlorination (ERD) from PCE-to-ethene (ETH). According to these laboratory results, which confirmed the presence of Dehalococcoides sp. and the vcrA gene, an in-situ ERD pilot test consisting of a single injection of lactate in a monitoring well was performed and monitored for 190 days. The parameters used to follow the performance of the ERD comprised the analysis of i) hydrochemistry, including redox potential (Eh), and the concentrations of redox sensitive species, chlorinated ethenes (CEs), lactate, and acetate; ii) stable isotope composition of carbon of CEs, and sulphur and oxygen of sulphate; and iii) 16S rRNA gene sequencing from groundwater samples. Thus, it was proved that the injection of lactate promoted sulphate-reducing conditions, with the subsequent decrease in Eh, which allowed for the full reductive dechlorination of PCE to ETH in the injection well. The biodegradation of CEs was also confirmed by the enrichment in 13C and carbon isotopic mass balances. The metagenomic results evidenced the shift in the composition of the microbial population towards the predominance of fermentative bacteria. Given the success of the in-situ pilot test, a full-scale ERD with lactate was then implemented at the site. After one year of treatment, PCE and trichloroethene were mostly depleted, whereas vinyl chloride (VC) and ETH were the predominant metabolites. Most importantly, the shift of the carbon isotopic mass balances towards more positive values confirmed the complete reductive dechlorination, including the VC-to-ETH reaction step. The combination of techniques used here provides complementary lines of evidence for the diagnosis of the intrinsic biodegradation potential of a polluted site, but also to monitor the progress, identify potential difficulties, and evaluate the success of ERD at the field scale.

Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain)

The bioremediation potential of an aquifer contaminated with tetrachloroethene (PCE) was assessed by combining hydrogeochemical data of the site, microcosm studies, metabolites concentrations, compound specific-stable carbon isotope analysis and the identification of selected reductive dechlorination biomarker genes. The characterization of the site through 10 monitoring wells evidenced that leaked PCE was transformed to TCE and cis-DCE via hydrogenolysis. Carbon isotopic mass balance of chlorinated ethenes pointed to two distinct sources of contamination and discarded relevant alternate degradation pathways in the aquifer. Application of specific-genus primers targeting Dehalococcoides mccartyi species and the vinyl chloride-to-ethene reductive dehalogenase vcrA indicated the presence of autochthonous bacteria capable of the complete dechlorination of PCE. The observed cis-DCE stall was consistent with the aquifer geochemistry (positive redox potentials; presence of dissolved oxygen, nitrate, and sulphate; absence of ferrous iron), which was thermodynamically favourable to dechlorinate highly chlorinated ethenes but required lower redox potentials to evolve beyond cis-DCE to the innocuous end product ethene. Accordingly, the addition of lactate or a mixture of ethanol plus methanol as electron donor sources in parallel field-derived anoxic microcosms accelerated dechlorination of PCE and passed cis-DCE up to ethene, unlike the controls (without amendments, representative of field natural attenuation). Lactate fermentation produced acetate at near-stoichiometric amounts. The array of techniques used in this study provided complementary lines of evidence to suggest that enhanced anaerobic bioremediation using lactate as electron donor source is a feasible strategy to successfully decontaminate this site.

Enrichment and characterization of hydrocarbon-degrading bacteria from petroleum refinery waste as potent bioaugmentation agent for in situ bioremediation

Intrinsic biodegradation potential of bacteria from petroleum refinery waste was investigated through isolation of cultivable strains and their characterization. Pseudomonas and Bacillus spp. populated the normal cultivable taxa while prolonged enrichment with hydrocarbons and crude oil yielded hydrocarbonoclastic bacteria of genera Burkholderia, Enterobacter, Kocuria, Pandoraea, etc. Strains isolated through enrichment showed assemblages of superior metabolic properties: utilization of aliphatic (C6-C22) and polyaromatic compounds, anaerobic growth with multiple terminal electron acceptors and higher biosurfactant production. Biodegradation of dodecane was studied thoroughly by GC–MS along with detection of gene encoding alkane hydroxylase (alkB). Microcosms bioaugmented with Enterobacter, Pandoraea and Burkholderia strains showed efficient biodegradation (98% TPH removal) well fitted in first order kinetic model with low rate constants and decreased half-life. This study proves that catabolically efficient bacteria resides naturally in complex petroleum refinery wastes and those can be useful for bioaugmentation based bioremediation.

A Multiple Lines of Evidence Framework to Evaluate Intrinsic Biodegradation of 1,4‐Dioxane

Development of a multiple lines of evidence (MLOE) framework to evaluate the intrinsic biodegradation potential of 1,4‐dioxane is vital to implementing management strategies at groundwater sites impacted by 1,4‐dioxane. A comprehensive MLOE approach was formed to provide significant evidence of natural degradation of 1,4‐dioxane comingled with tetrahydrofuran (THF) within a large, diffuse plume. State‐of‐the art molecular biological analyses and compound‐specific isotope analysis (CSIA) were employed to support more traditional approaches for data analysis (concentration trend analyses, spatial distribution, temporal changes, geochemical biodegradation attenuation indicators, plume mass estimates, and fate and transport modeling). The molecular analyses demonstrated that microorganisms capable of both metabolic and cometabolic degradation of 1,4‐dioxane were present throughout the groundwater plume, whereas the CSIA data provided supporting evidence of biodegradation. 1,4‐Dioxane biomarkers were present and abundant throughout the 1,4‐dioxane plume, and our biomarkers tracked the plume with reasonable accuracy. Evidence also suggests that THF‐driven cometabolic biodegradation as well as catabolic 1,4‐dioxane biodegradation were active at this site. These data supplemented the traditional lines of evidence approaches, which demonstrated that 1,4‐dioxane attenuation was occurring across the groundwater plume and that nondestructive physical processes alone did not account for the observed 1,4‐dioxane attenuation. This MLOE framework combining new and traditional analyses demonstrates that this site has a significant capacity for intrinsic biodegradation of 1,4‐dioxane. ©2016 Wiley Periodicals, Inc.

English

Biodegradation is the chemical breakdown of materials by a physiological environment. The term is often used in relation to ecology, waste management and environmental remediation. A specific gene from bacteria is identified and sequenced which has the capacity of oil degradation without any obvious strain specific discrimination using a combination of PCR and hybridization. The parameters of biodegradation that is culturing and PCR technique provide useful information for an assessment of the intrinsic biodegradation potential that is present at a site. PCR amplification products in the plasmid DNA of Pseudomonas aeruginosa was transformed into competent Escherichia coli cells. Thus the E. coli cells were conferred with oil degrading property and this was confirmed by growing them in Bushnell Hass medium along with petroleum oil. The E. coli cells were found to be catabolizing the oil. Results show that the capability for alkane degradation is a common trait in microbial communities. The method can be a very useful tool for the fast estimation of the biodegradation potential at polluted sites.   Key words: Detection, hydrocarbon biodegradation, PCR-hybridization, transformation.

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.

Intrinsic biodegradation potential of aromatic hydrocarbons in an alluvial aquifer – Potentials and limits of signature metabolite analysis and two stable isotope-based techniques

Three independent techniques were used to assess the biodegradation of monoaromatic hydrocarbons and low-molecular weight polyaromatic hydrocarbons in the alluvial aquifer at the site of a former cokery (Flémalle, Belgium).Firstly, a stable carbon isotope-based field method allowed quantifying biodegradation of monoaromatic compounds in situ and confirmed the degradation of naphthalene. No evidence could be deduced from stable isotope shifts for the intrinsic biodegradation of larger molecules such as methylnaphthalenes or acenaphthene. Secondly, using signature metabolite analysis, various intermediates of the anaerobic degradation of (poly-) aromatic and heterocyclic compounds were identified. The discovery of a novel metabolite of acenaphthene in groundwater samples permitted deeper insights into the anaerobic biodegradation of almost persistent environmental contaminants. A third method, microcosm incubations with 13C-labeled compounds under in situ-like conditions, complemented techniques one and two by providing quantitative information on contaminant biodegradation independent of molecule size and sorption properties. Thanks to stable isotope labels, the sensitivity of this method was much higher compared to classical microcosm studies. The 13C-microcosm approach allowed the determination of first-order rate constants for 13C-labeled benzene, naphthalene, or acenaphthene even in cases when degradation activities were only small. The plausibility of the third method was checked by comparing 13C-microcosm-derived rates to field-derived rates of the first approach. Further advantage of the use of 13C-labels in microcosms is that novel metabolites can be linked more easily to specific mother compounds even in complex systems. This was achieved using alluvial sediments where 13C-acenaphthyl methylsuccinate was identified as transformation product of the anaerobic degradation of acenaphthene.

Soil Physical Constraints on Intrinsic Biodegradation of Petroleum Vapors in a Layered Subsurface

Naturally occurring biodegradation of petroleum hydrocarbons in the vadose zone depends on the physical soil environment influencing field-scale gas exchange and pore-scale microbial metabolism. In this study, we evaluated the effect of soil physical heterogeneity on biodegradation of petroleum vapors in a 16-m-deep, layered vadose zone. Soil slurry experiments (soil/water ratio 10:30 w/w, 25°C) on benzene biodegradation under aerobic and well-mixed conditions indicated that the biodegradation potential in different textured soil samples was related to soil type rather than depth, in the order: sandy loam > fine sand > limestone. Similarly, O(2) consumption rates during in situ respiration tests performed at the site were higher in the sandy loam than in the fine sand, although the difference was less significant than in the slurries. Laboratory and field data generally agreed well and suggested a significant potential for aerobic biodegradation, even with nutrient-poor and deep subsurface conditions. In slurries of the sandy loam, the biodegradation potential declined with increasing in situ water saturation (i.e., decreasing air-filled porosity in the field). This showed a relation between antecedent undisturbed field conditions and the slurry biodegradation potential, and suggested airfilled porosity to be a key factor for the intrinsic biodegradation potential in the field.

Functional genes reveal the intrinsic PAH biodegradation potential in creosote-contaminated groundwater following in situ biostimulation

A small-scale functional gene array containing 15 functional gene probes targeting aliphatic and aromatic hydrocarbon biodegradation pathways was used to investigate the effect of a pilot-scale air sparging and nutrient infiltration treatment on hydrocarbon biodegradation in creosote-contaminated groundwater. Genes involved in the different phases of polycyclic aromatic hydrocarbon (PAH) biodegradation were detected with the functional gene array in the contaminant plume, thus indicating the presence of intrinsic biodegradation potential. However, the low aerobic fluorescein diacetate hydrolysis, the polymerase chain reaction (PCR) amplification of 16S rRNA genes closely similar to sulphate-reducing and denitrifying bacteria and the negligible decrease in contaminant concentrations showed that aerobic PAH biodegradation was limited in the anoxic groundwater. Increased abundance of PAH biodegradation genes was detected by functional gene array in the monitoring well located at the rear end of the biostimulated area, which indicated that air sparging and nutrient infiltration enhanced the intrinsic, aerobic PAH biodegradation. Furthermore, ten times higher naphthalene dioxygenase gene copy numbers were detected by real-time PCR in the biostimulated area, which was in good agreement with the functional gene array data. As a result, functional gene array analysis was demonstrated to provide a potential tool for evaluating the efficiency of the bioremediation treatment for enhancing hydrocarbon biodegradation in field-scale applications.

A new method for the detection of alkane-monooxygenase homologous genes ( alkB) in soils based on PCR-hybridization

An improved method was developed that allowed the specific detection of the gene alkB (coding for the rubredoxin dependent alkane monooxygenase) from bacteria without any obvious strain specific discrimination using a combination of PCR and hybridization. This approach enabled a fast culture-independent monitoring of environmental samples for the occurrence of alkB, and an estimation of the gene copy number and the genetic diversity. Both parameters provide useful informations for an assessment of the intrinsic biodegradation potential that is present at a site. The method was applied to soil samples from different uncontaminated sites. alkB was highly abundant and redundant in all soils tested. Potential biodegradation of n-alkanes was also demonstrated for these soils with substrate utilization assays. Cell numbers of hydrocarbon degraders estimated as MPN varied from 10 3 to 10 6 g − 1 soil (dry weight) for the different soils. Gene copy numbers estimated with MPN-PCR ranged within 1–40 * 10 4 ng − 1 soil DNA. Analysis of the diversity of the alkB sequences obtained from a grassland and an agricultural soil indicated that the alkane degrading microbial populations occurring at these sites were rather diverse. Compared on protein level, three major clusters were distinguishable for both soils that showed highest similarities to AlkB from the Gram-positives Nocardioides and Mycobacterium, and the Gram-negative Alcanivorax. The majority of the cloned AlkB sequences were homologous to proteins from the Gram-positive bacteria. However, significant differences from published sequences were observed; homologies varied from 50% to 90% (identity of amino acids).

Biodegradation of Chlorinated Solvents in Bioreactor Landfills

The objective of this research is to investigate the intrinsic biodegradation potential of the municipal solid waste (MSW) landfill ecosystem for chlorinated aliphatic hydrocarbons (CAHs). The research was conducted in two complementary systems: simulated landfill bioreactors and batch degradation experiment in serum bottles. Refuse samples excavated from a MSW landfill were tested in laboratory bioreactors operated with leachate recirculation and gas collection. Tetrachloroethene (PCE) and trichloroethene (TCE) were added to reactors and maintained at 20 μM each in leachate to simulate the long-term exposure of refuse microorganisms to CAHs in landfills. Anaerobic microbial cultures were derived from reactors at two different stages of refuse decomposition: active refuse decomposition stage representing young landfills and maturation phase representing aged landfills. These cultures were tested in serum bottles for their abilities to biodegrade target CAHs. Results of this study show that MSW landfills have an intrinsic reductive dechlorination capacity for PCE and TCE. The decomposition of refuse also enhanced reductive dechlorination in this study. This research suggests the potential to develop engineering strategies to promote chlorinated solvent degradation concurrently with refuse decomposition in MSW landfills.