Improving the anaerobic digestion of sewage sludge by adding cobalt nanoparticles.

This work evaluated the effects of cobalt nanoparticles (CoNPs) (0.025-7 mg/gVS) on the intensification of sewage sludge anaerobic digestion (AD) using biochemical methane potential (BMP) tests. This study was motivated by the need to improve the efficiency and stability of anaerobic digestion of sewage sludge, a critical process in waste management and renewable energy production. The effects at doses less than 2 mg/gVS were not substantial, but 3-7 mg/gVS improved the performance. The maximum biogas yield was 232 mL/gVS (at a dose of 7 mg/gVS), whereas it was 132 mL/gVS in the control (zero dose). Similarly, the reductions in the volatile solids and methane contents reached maxima of 16 and 74.3%, respectively. The analyses of volatile fatty acids, redox potential, and electron transfer system activity indicated that the addition of CoNPs stimulated the early stages of AD. Finally, acetate consumption and the increase in CH4 content suggested that CoNPs positively affected system stability and acetoclastic methanogenesis. That is, CoNPs effectively intensified the behaviour and stability of the anaerobic process. The novelty of this research lies in the comprehensive evaluation of the effects of CoNPs across a wide range of doses on sewage sludge AD, providing new insights into the optimisation of this process for increased biogas production and organic matter reduction.

Simulation of anaerobic biodegradation process in a tubular bioreactor with a biofilm layer: Steady-state and unsteady-state conditions.

In this paper, anaerobic biodegradation process in a tubular bioreactor with an inner biofilm layer for steady-state and unsteady-state conditions are simulated. The effects of various parameters including bioreactor diameter, fraction of active biomass transferred to liquid phase, and residence time of the liquid on bioreactor performance are examined. Simulations indicate that decreasing diameter of bioreactor leads to increasing degree of conversion of the substrate in liquid phase and decreasing dimensionless concentration of the substrate in biofilm. With an increase in the fraction of active biomass transferred to liquid, substrate concentrations in liquid and biofilm slightly vary. Increased residence time of the liquid phase results in the degree of conversion of substrate goes up, but substrate concentration in biofilm lowers a little. In addition, it is found that biomass concentration of liquid phase is boosted with decreased bioreactor diameter and increased residence time of liquid. A proportional-integral controller is designed and the tuned parameters of K P = - 0.131 and K I = 0.02 are obtained using genetic algorithm. It is observed that controller regulate well the degree of conversion of the substrate within 120 s for both servo and regulatory modes.

Biodegradation of Crude Oil by Nitrate-Reducing, Sulfate-Reducing, and Methanogenic Microbial Communities under High-Pressure Conditions.

Carbon capture, utilization, and storage (CCUS) is an important component in many national net-zero strategies, and ensuring that CO2 can be safely and economically stored in geological systems is critical. Recent discoveries have shown that microbial processes (e.g., methanogenesis) can modify fluid composition and fluid dynamics within the storage reservoir. Oil reservoirs are under high pressure, but the influence of pressure on the petroleum microbial community has been previously overlooked. To better understand microbial community dynamics in deep oil reservoirs, we designed an experiment to examine the effect of high pressure (12 megapascals [MPa], 60 °C) on nitrate-reducing, sulfate-reducing, and methanogenic enrichment cultures. Cultures were exposed to these conditions for 90 d and compared with a control exposed to atmospheric pressure (0.1 MPa, 60 °C). The degradation characteristic oil compounds were confirmed by thin-layer analysis of oil SARA (saturates, aromatics, resins, and asphaltenes) family component rods. We found that the asphaltene component in crude oil was biodegraded under high pressure, but the concentration of asphaltenes increased under atmospheric pressure. Gas chromatography analyses of saturates showed that short-chain saturates (C8-C12) were biodegraded under high and atmospheric pressure, especially in the methanogenic enrichment culture under high pressure (the ratio of change was -81%), resulting in an increased relative abundance of medium- and long-chain saturates. In the nitrate-reducing and sulfate-reducing enrichment cultures, long-chain saturates (C22-C32) were biodegraded in cultures exposed to high-pressure and anaerobic conditions, with a ratio of change of -8.0% and -2.3%, respectively. However, the relative proportion of long-chain saturates (C22-C32) increased under atmospheric pressure. Gas Chromatography Mass Spectrometry analyses of aromatics showed that several naphthalene series compounds (naphthalene, C1-naphthalene, and C2-naphthalene) were biodegraded in the sulfate-reducing enrichment under both atmospheric pressure and high pressure. Our study has discerned the linkages between the biodegradation characteristics of crude oil and pressures, which is important for the future application of bioenergy with CCUS (bio-CCUS).

Enrichment of Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Strictly Anaerobic Sulfate-Reducing Cultures from Contaminated Soil and Sediment.

Sulfate-reducing bacteria (SRB) are crucial players in global biogeochemical cycling and some have been implicated in the anaerobic biodegradation of organic pollutants, including recalcitrant and hazardous polycyclic aromatic hydrocarbons (PAHs). Obtaining PAH-degrading SRB cultures for laboratories is of paramount importance in the development of the young field of anaerobic biodegradation of PAHs. SRB grow exceptionally slowly on PAH substrates and are highly sensitive to oxygen. Consequently, enrichment and maintenance of PAH-degrading SRB cultures and characterization of the biodegradation process remain a tedious and formidable task, especially for new researchers. To address these technical constraints, we have developed robust and effective protocols for obtaining and characterizing PAH-degrading SRB cultures. In this set of protocols, we describe step-by-step procedures for preparing inocula from contaminated soil or sediment, preparing anoxic medium, establishing enrichment cultures with PAHs as substrates under completely anaerobic sulfate-reducing conditions, successive culture transfers to obtain highly enriched cultures, rapid verification of the viability of SRB in slow-growing cultures, assessment of PAH degradation by extracting residuals using organic solvent and subsequent analysis by gas chromatography-mass spectrometry, and spectrophotometric determination of sulfate and sulfide in miniaturized, medium-throughput format. These protocols are expected to serve as a comprehensive manual for obtaining and characterizing PAH-degrading sulfate-reducing cultures. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Obtaining PAH-degrading strictly anaerobic sulfate-reducing enrichment cultures from contaminated soil and sediment Support Protocol 1: Operation and maintenance of an anaerobic workstation Support Protocol 2: Setup of gas purging systems for preparing anoxic solutions Support Protocol 3: Verification of viability in slow-growing SRB enrichment cultures Support Protocol 4: Extraction of genomic DNA from low-biomass cultures Basic Protocol 2: Extraction of residual PAH from liquid culture and analysis by GC-MS Basic Protocol 3: Spectrophotometric determination of sulfate concentration in SRB cultures Basic Protocol 4: Spectrophotometric determination of sulfide concentrations in SRB cultures by the methylene blue method Alternate Protocol: Spectrophotometric determination of sulfide concentrations in SRB cultures by the colloidal copper sulfide method.

Meta-Analysis and Machine Learning Models for Anaerobic Biodegradation Rates of Organic Contaminants in Sediments and Sludge.

Anaerobic biodegradation rates (half-lives) of organic chemicals are pivotal for environmental risk assessment and remediation. Traditional experimental evaluation, constrained by prolonged, oxygen-free conditions, struggles to keep pace with emerging contaminants. Data-driven machine learning (ML) models serve as promising complements. However, reported quantitative structure-biodegradation relationships or ML models on anaerobic biodegradation are mostly based on small data sets (<100 records) and neglect experimental conditions, usually achieving compromised predictions. This work aimed to develop ML models for predicting the biodegradation half-lives of organic pollutants in anaerobic environments (i.e., sediment/soil and sludge). Focusing on important features of both chemicals and experimental conditions, we first curated two data sets, one for sediment/soil (SED) and the other for sludge (SLD), covering 978 records for 206 chemicals from the literature, and then conducted a meta-analysis. Next, we built a binary classification (half-life of 30 days as the cutoff) model with an accuracy of 81% and a regression model with R2 of 0.56 for SED based on LightGBM (80% and 0.31 for SLD based on Extra tree, respectively). The model interpretations underscored the significance of experimental conditions (e.g., temperature and inoculum dosage), as evidenced by their high feature importance, and the models were found to correctly capture the effects of chemical substructures, for example, branched structures and aromatic rings prolonged half-lives while methyl group and ortho-substitution on rings shortened half-lives. The applicability domains of the models were also defined, resulting in reasonable prediction for the half-lives of 41% (SED) or 67% (SLD) of over 4000 persistent, bioaccumulative, and toxic chemicals. Overall, this study pioneers ML models for predicting the anaerobic degradation half-lives, offering valuable support for future evaluation and implementation of chemical anaerobic biodegradation.

Anaerobic biodegradation enables zero liquid discharge of two-stage nanofiltration system for microelectronic wastewater treatment.

Nanofiltration (NF) is a promising technology in the treatment of microelectronic wastewater. However, the treatment of concentrate derived from NF system remains a substantial technical challenge, impeding the achievement of the zero liquid discharge (ZLD) goal in microelectronic wastewater industries. Herein, a ZLD system, coupling a two-stage NF technology with anaerobic biotechnology was proposed for the treatment of tetramethylammonium hydroxide (TMAH)-contained microelectronic wastewater. The two-stage NF system exhibited favorable efficacy in the removal of conductivity (96 %), total organic carbon (TOC, 90 %), and TMAH (96 %) from microelectronic wastewater. The membrane fouling of this system was dominated by organic fouling, with the second stage NF membrane experiencing a more serious fouling compared to the first stage membrane. The anaerobic biotechnology achieved a near-complete removal of TMAH and an 80 % reduction in TOC for the first stage NF concentrate. Methyloversatilis was the key genus involved in the anaerobic treatment of the microelectronic wastewater concentrate. Specific genes, including dmd-tmd, mtbA, mttB and mttC were identified as significant players in mediating the dehydrogenase and methyl transfer pathways during the process of TMAH biodegradation. This study highlights the potential of anaerobic biodegradation to achieve ZLD in the treatment of TMAH-contained microelectronic wastewater by NF system.

Molecular and microbial insights towards anaerobic biodegradation of anionic polyacrylamide in oil sands tailings.

Anionic polyacrylamide (A-PAM) is widely used as a flocculant in the management of oil sands tailings. Nevertheless, apprehensions arise regarding its potential biodegradation and environmental consequences within the context of oil sands tailings. Consequently, it is imperative to delve into the anaerobic biodegradation of A-PAM in oil sands tailings to gain a comprehensive understanding of its influence on tailings water quality. This work explored the dynamics of A-PAM biodegradation across concentrations: 50, 100, 250, 500, 1000, and 2000 mg/kg TS. The results showed a significant decrease in A-PAM concentration and molecular weight at lower concentrations (50 and 100 mg/kg TS) compared to higher ones, suggesting enhanced degradation efficiency. Likewise, the organic transformation and methane production exhibited dependency on A-PAM concentrations. The peak concentrations observed were 20.0 mg/L for volatile fatty acids (VFAs), 0.07 mg/L for acrylamide (AMD), and 8.9 mL for methane yield, with these maxima being recorded at 50 mg/kg TS. The biodegradation efficiency diminishes at higher concentrations of A-PAM, potentially due to the inhibitory effects of polyacrylic acid accumulation. A-PAM biodegradation under anaerobic condition did not contribute to acute toxicity or genotoxicity. SEM-EDS, FT-IR and XRD analyses further revealed that higher concentrations of A-PAM inhibited the biodegradation by altering floc structure and composition, thereby restricting the microbial activity. Major microorganisms, including Smithella, Candidatus_Cloacimonas, W5, XBB1006, and DMER64 were identified, highlighting A-PAM's dual role as a source of carbon and nitrogen under anaerobic conditions. The above findings from this research not only significantly advance understanding of A-PAM's environmental behavior but also contribute to the effective management practices in oil sands tailings.

Effects of Fe/Fe-Mn oxides loaded biochar on anaerobic degradation of typical phenolic compounds in coal gasification wastewater: Performance and mechanism.

In this study, two kinds of magnetic biochar (BC) were synthesized by loading Fe (FeBC) and Fe-Mn oxides (FMBC) and their effects on anaerobic phenolics degradation were investigated. Compared with BC/FMBC, FeBC addition achieved the superior phenolics biodegradation even for 3,5-xylenol. Compared with control, FeBC addition enhanced CH4 production by 100.1 % with the lag time shortened from 9.5 days to 6.6 days while it increased to 11.2 days with FMBC addition. FeBC addition activated adsorption-biodegradation and Fe (III) reduction with the improved electron transfer activity, adenosine triphosphate and cytochrome C concentrations. Abundant phenol degrading bacteria, electroactive bacteria, syntrophic partners could be enriched by FeBC addition, contributing to the enhanced benzoyl-CoA and methanogenesis pathways. However, this enhancement was inhibited by FMBC addition owing to the accumulation of reactive oxygen species. This study provided novel insights into the application of magnetic BC to enhanced anaerobic treatment of phenolic wastewater.

Effects of electron acceptors and donors on anaerobic biodegradation of PAHs in marine sediments.

Polycyclic aromatic hydrocarbons (PAHs) are typical organic pollutants accumulated in the environment. PAHs' bioremediation in sediments can be promoted by adding electron acceptor (EA) and electron donor (ED). Bicarbonate and sulfate were chosen as two EAs, and acetate and lactate were selected as two EDs. Six groups of amendments were added into the sediments to access their role in the anaerobic biodegradation of five PAHs, containing phenanthrene, anthracene, fluoranthene, pyrene, and benzo[a]pyrene. The concentrations of PAHs, EAs and EDs, electron transport system activity, and microbial diversity were analyzed during 126-day biodegradation in serum bottles. The HA group (bicarbonate and acetate) achieved the maximum PAH degradation efficiency of 89.67 %, followed by the SL group (sulfate and lactate) with 87.10 %. As the main PAHs degrading bacteria, the abundance of Marinobacter in H group was 8.62 %, and the addition of acetate significantly increased the abundance of Marinobacter in the HA group by 75.65 %.

Strategy for Remediation of Polychlorinated Biphenyls-Contaminated Soil Through Redox Management Based on Electronegativity of the Contaminants.

Literature review reveals that Persistent Organic Pollutants (POPs), such as polychlorinated biphenyls (PCBs), are electron deficient compounds due to the presence of highly electronegative groups. Hence, they are more amenable to anaerobic biodegradation rather than oxidative metabolism. However, the studies on PCBs bioremediation are more inclined towards aerobic treatment. Besides, the past studies are mainly centered on screening and application of PCB-degrading microorganisms. In our opinion the degradative capacity is already present in the native microflora, and choice of electron donor is of paramount importance for faster reductive metabolism of PCBs. In this study, the use of methanol as electron donor with cow dung as the general microbial inoculum resulted in high specific rate of degradation (0.0542-0.0637 /day) for high-chlorinated biphenyls. The % removal of PCBs ranged between 67.7 and 71.7%. It may be the first study on the application of methanol as a cheap electron donor for PCBs biodegradation without bioaugmentation with specifically selected microorganisms.

Anaerobic biodegradation of pyrene and benzo[a]pyrene by a new sulfate-reducing Desulforamulus aquiferis strain DSA.

The study of anaerobic high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) biodegradation under sulfate-reducing conditions by microorganisms, including microbial species responsible for biodegradation and relative metabolic processes, remains in its infancy. Here, we found that a new sulfate-reducer, designated as Desulforamulus aquiferis strain DSA, could biodegrade pyrene and benzo[a]pyrene (two kinds of HMW-PAHs) coupled with the reduction of sulfate to sulfide. Interestingly, strain DSA could simultaneously biodegrade pyrene and benzo[a]pyrene when they co-existed in culture. Additionally, the metabolic processes for anaerobic pyrene and benzo[a]pyrene biodegradation by strain DSA were newly proposed in this study based on the detection of intermediates, quantum chemical calculations and analyses of the genome and RTqPCR. The initial activation step for anaerobic pyrene and benzo[a]pyrene biodegradation by strain DSA was identified as the formation of pyrene-2-carboxylic acid and benzo[a]pyrene-11-carboxylic acid by carboxylation Thereafter, CoA ligase, ring reduction through hydrogenation, and ring cracking occurred, and short-chain fatty acids and carbon dioxide were identified as the final products. Additionally, DSA could also utilize benzene, naphthalene, anthracene, phenanthrene, and benz[a]anthracene as carbon sources. Our study can provide new guidance for the anaerobic HMW-PAHs biodegradation under sulfate-reducing conditions.

Assessing labelled carbon assimilation from poly butylene adipate-co-terephthalate (PBAT) monomers during thermophilic anaerobic digestion.

PBAT (poly butylene adipate-co-terephthalate) is a widely used biodegradable plastic, but the knowledge about its metabolization in anaerobic environments is very limited. In this study, the anaerobic digester sludge from a municipal wastewater treatment plant was used as inoculum to investigate the biodegradability of PBAT monomers in thermophilic conditions. The research employs a combination of 13C-labelled monomers and proteogenomics to track the labelled carbon and identify the microorganisms involved. A total of 122 labelled peptides of interest were identified for adipic acid (AA) and 1,4-butanedio (BD). Through the time-dependent isotopic enrichment and isotopic profile distributions, Bacteroides, Ichthyobacterium, and Methanosarcina were proven to be directly involved in the metabolization of at least one monomer. This study provides a first insight into the identity and genomic potential of microorganisms responsible for biodegradability of PBAT monomers during anaerobic digestion under thermophilic conditions.

Anaerobic biodegradation of phenanthrene and pyrene by sulfate-reducing cultures enriched from contaminated freshwater lake sediments.

Our current understanding of the susceptibility of hazardous polycyclic aromatic hydrocarbons (PAHs) to anaerobic microbial degradation is very limited. In the present study, we obtained phenanthrene- and pyrene-degrading strictly anaerobic sulfate-reducing enrichments using contaminated freshwater lake sediments as the source material. The highly enriched phenanthrene-degrading culture, MMKS23, was dominated (98%) by a sulfate-reducing bacterium belonging to the genus Desulfovibrio. While Desulfovibrio sp. was also predominant (79%) in the pyrene-degrading enrichment culture, MMKS44, an anoxygenic purple non-sulfur bacterium, Rhodopseudomonas sp., constituted a significant fraction (18%) of the total microbial community. Phenanthrene or pyrene biodegradation by the enrichment cultures was coupled with sulfate reduction, as evident from near stoichiometric consumption of sulfate and accumulation of sulfide. Also, there was almost complete inhibition of substrate degradation in the presence of an inhibitor of sulfate reduction, i.e., 20 mM MoO42-, in the culture medium. After 180 days of incubation, about 79.40 µM phenanthrene was degraded in the MMKS23 culture, resulting in the consumption of 806.80 µM sulfate and accumulation of 625.80 µM sulfide. Anaerobic pyrene biodegradation by the MMKS44 culture was relatively slow. About 22.30 µM of the substrate was degraded after 180 days resulting in the depletion of 239 µM sulfate and accumulation of 196.90 µM sulfide. Biodegradation of phenanthrene by the enrichment yielded a metabolite, phenanthrene-2-carboxylic acid, suggesting that carboxylation could be a widespread initial step of phenanthrene activation under sulfate-reducing conditions. Overall, this novel study demonstrates the ability of sulfate-reducing bacteria (SRB), dwelling in contaminated freshwater sediments to anaerobically biodegrade three-ringed phenanthrene and highly recalcitrant four-ringed pyrene. Our findings suggest that SRB could play a crucial role in the natural attenuation of PAHs in anoxic freshwater sediments.

Biodegradation of 2-methylpentane in fluid fine tailings amended with a mixture of iso-alkanes under sulfate-reducing conditions.

Anaerobic microorganisms in Canada Natural Upgrading Limited (CNUL) fluid fine tailings (FFT) are sustained by residual solvent hydrocarbons. Although FFT are methanogenic in nature, sulfate-reducing microorganisms represent a significant portion of FFT bacterial community. In this study, we examined biodegradation of three iso-alkanes (2-methylbutane, 2-methylpentane, and 3-methylpentane), representing major iso-alkanes in paraffinic solvent, in CNUL FFT under sulfate-reducing conditions. During ∼1100 days of incubation, only 2-methylpentane was degraded partially, whereas 2-methylbutane and 3-methylpentane were not degraded. During active degradation of 2-methylpentane, the bacterial community was dominated by Anaerolineaceae followed by Syntrophaceae, Peptococcaceae, Desulfobacteraceae, and Desulfobulbaceae. The archaeal community was co-dominated by acetoclastic (Methanosaetaceae) and hydrogenotrophic (Methanobacteriaceae) methanogens. This study underlines the limited capability of the microbial community indigenous to CNUL FFT in degrading recalcitrant iso-alkanes under sulfate-reducing conditions.

Sequentially modified carbon felt for enhanced p-nitrophenol biodegradation through direct interspecific electron transfer.

The widely applied aromatic nitration in modern industry leads to toxic p-nitrophenol (PNP) in environment. Exploring its efficient degradation routes is of great interests. In this study, a novel four-step sequential modification procedure was developed to increase the specific surface area, functional group, hydrophilicity, and conductivity of carbon felt (CF). The implementation of the modified CF promoted reductive PNP biodegradation, attaining 95.2 ± 0.8% of removal efficiency with less accumulation of highly toxic organic intermediates (e.g., p-aminophenol), compared to carrier-free and CF-packed biosystems. The constructed anaerobic-aerobic process with modified CF in 219-d continuous operation achieved further removal of carbon and nitrogen containing intermediates and partial mineralization of PNP. The modified CF promoted the secretion of extracellular polymeric substances (EPS) and cytochrome c (Cyt c), which were essential components to facilitate direct interspecies electron transfer (DIET). Synergistic relationship was deduced that glucose was converted into volatile fatty acids by fermenters (e.g., Longilinea and Syntrophobacter), which donated electrons to the PNP degraders (e.g., Bacteroidetes_vadinHA17) through DIET channels (CF, Cyt c, EPS) to complete PNP removal. This study proposes a novel strategy using engineered conductive material to enhance the DIET process for efficient and sustainable PNP bioremediation.

CO2 improves the anaerobic biodegradation intensity and selectivity of heterocyclic hydrocarbons in heavy oil.

Heterocyclic hydrocarbons pollution generated by oil spills and oilfield wastewater discharges threatens the ecological environment and human health. Here we described a strategy that combines the greenhouse gas CO2 reduction with microbial remediation. In the presence of nitrate, CO2 can improve the biodegradation efficiency of the resins and asphaltenes in heavy oil, particularly the biodegradation selectivity of the polar heterocyclic compounds by the newly isolated Klebsiella michiganensis. This strain encoded 80 genes for the xenobiotic biodegradation and metabolism, and can efficiently utilize CO2 when degrading heavy oil. The total abundance of resins and asphaltenes decreased significantly with CO2, from 40.816% to 26.909%, to 28.873% with O2, and to 36.985% with N2. The transcripts per million (TPM) value of accA gene was 57.81 under CO2 condition, while respectively 8.86 and 21.23 under O2 and N2 conditions. Under CO2 condition, the total relative percentage of N1-type heterocyclic compounds was selectively decreased from 32.25% to 22.78%, resulting in the heavy oil viscosity decreased by 46.29%. These results demonstrated a novel anaerobic degradation mechanism that CO2 can promote the anaerobic biodegradation of heterocyclic hydrocarbons in heavy oil, which provides a promising biotreatment technology for the oil-contaminated water.

Anaerobic biodegradation of polycyclic aromatic hydrocarbons.

Although many anaerobic microorganisms that can degrade PAHs have been harnessed, there is still a large gap between laboratory achievements and practical applications. Here, we review the recent advances in the biodegradation of PAHs under anoxic conditions and highlight the mechanistic insights into the metabolic pathways and functional genes. Achievements of practical application and enhancing strategies of anaerobic PAHs bioremediation in soil were summarized. Based on the concerned issues during research, perspectives of further development were proposed including time-consuming enrichment, byproducts with unknown toxicity, and activity inhibition with low temperatures. In addition, meta-omics, synthetic biology and engineering microbiome of developing microbial inoculum for anaerobic bioremediation applications are discussed. We anticipate that integrating the theoretical research on PAHs anaerobic biodegradation and its successful application will advance the development of anaerobic bioremediation.

Anaerobic Degradation of Naphthalene and Pyrene by Sulfate-Reducing Cultures Enriched from Former Manufactured Gas Plant Soil.

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) under completely anaerobic sulfate-reducing conditions is an energetically challenging process. To date, anaerobic degradations of only two-ringed naphthalene and three-ringed phenanthrene by sediment-free and enriched sulfate-reducing bacteria have been reported. In this study, sulfate-reducing enrichment cultures capable of degrading naphthalene and four-ringed PAH, pyrene, were enriched from a contaminated former gas plant site soil. Bacterial community composition analysis revealed that a naphthalene-degrading enrichment culture, MMNap, was dominated (84.90%) by a Gram-positive endospore-forming member of the genus Desulfotomaculum with minor contribution (8.60%) from a member of Clostridium. The pyrene-degrading enrichment, MMPyr, was dominated (97.40%) by a species of Desulfotomaculum. The sequences representing the Desulfotomaculum phylotypes shared 98.80% similarity to each other. After 150 days of incubation, MMNap degraded 195 µM naphthalene with simultaneous reduction of sulfate and accumulation of sulfide. Similarly, MMPyr degraded 114 µM pyrene during 180 days of incubation with nearly stochiometric sulfate consumption and sulfide accumulation. In both cases, the addition of sulfate reduction inhibitor, molybdate (20 mM), resulted in complete cessation of the substrate utilization and sulfate reduction that clearly indicated the major role of the sulfate-reducing Desulfotomaculum in biodegradation of the two PAHs. This study is the first report on anaerobic pyrene degradation by a matrix-free, strictly anaerobic, and sulfate-reducing enrichment culture.

Oriented bio-feeding control of the anaerobic biodegradation of ethinyl estradiol.

Up to 95% of hormones are excreted into domestic wastewater with urine or feces, but their macromolecules are difficult to biodegrade. This project studies the treatment of Ethinyl Estradiol (EE2) in swine wastewater in an Upstream Solids Reactor (USR), and explores a new method for oriented bio-feeding to regulate the anaerobic biodegradation process. It was found that the metabolism of lactic acid and propionic acid was accompanied by changes in EE2 content, but lactic acid molecules were not readily bioavailable, so adding propionic acid was more suitable. However, controlling the pH to lower (4.73) and higher (8.73) values inhibited further fermentation of acetic acid and propionic acid, which was not favorable for the removal of EE2. This is simply due to the fact that propionic acid as a carbon source changes the preference of the microbes for consuming EE2. The order of the effect of addition of propionic acid on the removal of EE2 was as follows: P400>P800>P0>P200 (addition of propionic acid). The P400 removal efficiency increased from 60% to 85%. In the metabolism of EE2, after oxidation, hydrolysis, ketosis, hydroxylation and enzymatic action, dienoic acid and oleic acid were generated, and there was no secondary pollution from EE2 metabolites. In conclusion, feeding microorganisms with propionic acid can enhance the anaerobic biodegradation of EE2, providing a new strategy for the anaerobic biodegradation and bioremediation of refractory pollutants.

Anaerobic phenanthrene biodegradation by a new salt-tolerant/halophilic and nitrate-reducing Virgibacillus halodenitrificans strain PheN4 and metabolic processes exploration.

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) under hypersaline environments has received increasing attention, whereas the study of anaerobic PAH biodegradation under hypersaline environments is still lacking. Here, we found a pure culture designated PheN4, which was affiliated with Virgibacillus halodenitrificans and could degrade phenanthrene with nitrate as the terminal electron acceptor and a wide range of salinities (from 0.3% to 20%) under anaerobic environments. The optimal salinity for biodegradation of phenanthrene by PheN4 was 5%, which could degrade 93.5% of 0.62 ± 0.04 mM phenanthrene within 10 days with the initial inoculum of 0.01 gVSS/L. Meanwhile, an increased microbial amount could efficiently promote the phenanthrene biodegradation rate. The metabolic processes of anaerobic phenanthrene biodegradation under hypersaline conditions by PheN4 were proposed based on intermediates and genome analyses. Phenanthrene was initially activated via methylation to form 2-methylphenanthrene. Next, fumarate addition and ß-oxidation or direct oxidation of the methyl group, ring reduction and ring cleavage were identified as the midstream and downstream steps. In addition, PheN4 could utilize benzene, naphthalene, and anthracene as carbon sources, but Benz[a]anthracene, pyrene, and Benzo[a]pyrene could not be biodegraded by PheN4. This study could provide some guidance for the bioremediation of PAH pollutants in anaerobic and hypersaline zones.