Nitrate-reducing Conditions Research Articles

Physiological response of Desulfurispirillum indicum S5 to arsenate and nitrate as terminal electron acceptors

The ability of anaerobic prokaryotes to employ different terminal electron acceptors for respiration enables these organisms to flourish in subsurface ecosystems. Desulfurispirillum indicum strain S5 is an obligate anaerobic bacterium that is able to grow by respiring a range of different electron acceptors, including arsenate and nitrate. Here, we examined the growth, electron acceptor utilization, and gene expression of D. indicum growing under arsenate and nitrate-reducing conditions. Consistent with thermodynamic predictions, the experimental results showed that the reduction of nitrate to ammonium yielded higher cell densities than the reduction of arsenate to arsenite. However, D. indicum grew considerably faster by respiration on arsenate compared with nitrate, with doubling times of 4.3 ± 0.2 h and 19.2 ± 2.0 h, respectively. Desulfurispirillum indicum growing on both electron acceptors exhibited the preferential utilization of arsenate before nitrate. The expression of the arsenate reductase gene arrA was up-regulated approximately 100-fold during arsenate reduction, as determined by qRT-PCR. Conversely, the nitrate reductase genes narG and napA were not differentially regulated under the conditions tested. The results of this study suggest that physiology, rather than thermodynamics, controls the growth rates and hierarchy of electron acceptor utilization in D. indicum.

Physiological and Proteomic Adaptation of “Aromatoleum aromaticum” EbN1 to Low Growth Rates in Benzoate-Limited, Anoxic Chemostats

"Aromatoleum aromaticum" EbN1 was cultivated at different growth rates in benzoate-limited chemostats under nitrate-reducing conditions. Physiological characteristics, proteome dynamics, phospholipid-linked fatty acid (PLFA) composition, and poly(3-hydroxybutyrate) (PHB) content were analyzed in steady-state cells at low (μ(low)) (0.036 h(-1)), medium (μ(med)) (0.108 h(-1)), and high (μ(high)) (0.180 h(-1)) growth rates. A positive correlation to growth rate was observed for cellular parameters (cell size, and DNA and protein contents). The free energy consumed for biomass formation steadily increased with growth rate. In contrast, the energy demand for maintenance increased only from μ(low) to μ(med) and then remained constant until μ(high). The most comprehensive proteomic changes were observed at μ(low) compared to μ(high). Uniformly decreased abundances of protein components of the anaerobic benzoyl coenzyme A (benzoyl-CoA) pathway, central carbon metabolism, and information processing agree with a general deceleration of benzoate metabolism and cellular processes in response to slow growth. In contrast, increased abundances were observed at μ(low) for diverse catabolic proteins and components of uptake systems in the absence of the respective substrate (aromatic or aliphatic compounds) and for proteins involved in stress responses. This potential catabolic versatility and stress defense during slow growth may be interpreted as preparation for future needs.

Biodegradation of Crude Oil from the BP Oil Spill in the Marsh Sediments of Southeast Louisiana, USA

The significant challenges presented by the April 20, 2010 explosion, sinking, and subsequent oil spill of the Deepwater Horizon drilling platform in Canyon Block 252 about 52 miles southeast of Venice, LA, USA greatly impacted Louisiana's coastal ecosystem including the sea food industry, recreational fishing, and tourism. The short-term and long-term impact of this oil spill are significant, and the Deepwater Horizon spill is potentially both an economic and an ecological disaster. Microbes present in the water column and sediments have the potential to degrade the oil. Oil degradation could be enhanced by biostimulation method. The conventional approach to bioremediation of petroleum hydrocarbon is based on aerobic processes. Anaerobic bioremediation has been tested only in a very few cases and is still considered experimental. The currently practiced conventional in situ biorestoration of petroleum-contaminated soils and ground water relies on the supply of oxygen to the subsurface to enhance natural aerobic processes to remediate the contaminants. However, anaerobic microbial processes can be significant in oxygen-depleted subsurface environments and sediments that are contaminated with petroleum-based compounds such as oil-impacted marshes in Louisiana. The goal of this work was to identify the right conditions for the indigenous anaerobic bacteria present in the contaminated sites to enhance degradation of petroleum hydrocarbons. We evaluated the ability of microorganisms under a variety of electron acceptor conditions to degrade petroleum hydrocarbons. Researched microbial systems include sulfate-, nitrate-reducing bacteria, and fermenting bacteria. The results indicated that anaerobic bacteria are viable candidates for bioremediation. Enhanced biodegradation was attained under mixed electron acceptor conditions, where various electron-accepting anaerobes coexisted and aided in degrading complex petroleum hydrocarbon components of marsh sediments in the coastal Louisiana. Significant degradation of oil also occurred under sulfate-reducing and nitrate-reducing conditions.

Biodegradation of rhamnolipids in liquid cultures: Effect of biosurfactant dissipation on diesel fuel/B20 blend biodegradation efficiency and bacterial community composition

Bacterial utilization of rhamnolipids during biosurfactant-supplemented biodegradation of diesel and B20 (20% biodiesel and 80% diesel v/v) fuels was evaluated under conditions with full aeration or with nitrate and nitrite as electron acceptors. Rhamnolipid-induced changes in community dynamics were assessed by employing real-time PCR and the ddCt method for relative quantification.The experiments with rhamnolipids at 150mg/l, approx. double critical micelle concentration (CMC) and diesel oil confirmed that rhamnolipids were readily degraded by a soil-isolated consortium of hydrocarbon degraders in all samples, under both aerobic and nitrate-reducing conditions. The presence of rhamnolipids increased the dissipation rates for B20 constituents under aerobic conditions, but did not influence the biodegradation rate of pure diesel. No effect was observed under nitrate-reducing conditions. The biodegradation of rhamnolipids did not favor the growth of any specific consortium member, which proved that the employed biosurfactant did not interfere with the microbial equilibrium during diesel/biodiesel biodegradation.

Time and cost efficient biodegradation of diesel in a continuous-upflow packed bed biofilm reactor and effect of surfactant GAELE

BACKGROUND: Biodegradation of diesel hydrocarbons using bioreactors has been proposed as an alternative for diesel contaminated sites remediation. To make this alternative feasible, several factors must be optimized or improved: reducing hydraulic retention times (HRT) and applying design methods to enhance the access of the microorganisms to low soluble and recalcitrant compounds like hydrocarbon fuels. In the present work a time and cost efficient continuous-flow packed bed bioreactor at low HRT was designed and evaluated. The effect of non-previously studied anionic surfactant GAELE (glycolic acid ethoxylate lauryl ether) was also investigated. RESULTS: A continuous-upflow packed bed bioreactor (CPR) was built using an inexpensive support made of volcanic and alluvial stones. The biodegradation experiments conducted with a 12-month-old biofilm at a fixed HRT of 0.5 h, recorded removal of up to 97.9% at a diesel concentration of 1120 mg L−1 with GAELE. A first-order rate constant of 0.10 h−1 was calculated. Kinetic analysis using Arvin's model, which introduces mass transfer to the biofilm, showed statistical differences in the kinetic rate parameters (P < 0.001). Moreover, GAELE significantly increased biodegradability at high concentrations, with BOD5 and COD removals up to 90.8 and 80.7%, respectively. Putative hydrocarbon degrading bacteria responsible for the degradation under nitrate-reducing conditions were positively identified. CONCLUSIONS: The continuous-upflow packed bed reactor was capable of high percentage diesel biodegradation at short HRTs. The use of GAELE increased diesel availability and thus enhanced hydrocarbon removal. Therefore, CPR packed with volcanic and alluvial stones combined with GAELE showed potential for the remediation of diesel-impacted sites. Copyright © 2012 Society of Chemical Industry

Anaerobic degradation of 4-methylbenzoate by a newly isolated denitrifying bacterium, strain pMbN1.

A novel alphaproteobacterium isolated from freshwater sediments, strain pMbN1, degrades 4-methylbenzoate to CO(2) under nitrate-reducing conditions. While strain pMbN1 utilizes several benzoate derivatives and other polar aromatic compounds, it cannot degrade p-xylene or other hydrocarbons. Based on 16S rRNA gene sequence analysis, strain pMbN1 is affiliated with the genus Magnetospirillum.

Comparative survey of potential nitrate and sulfate reduction rates in aquatic sediments

Nitrate and sulfate are two major terminal electron acceptors of anaerobic respiration in nearshore sediments. Potential nitrate and sulfate reduction rates (NRR and SRR) were determined on surficial sediments sampled at 14 sites representing a wide range of shallow-water depositional environments. The rates were obtained by supplying undisturbed slices of sediments with nitrate, sulfate or both using a flow-through reactor technique. No external electron donor was added to the sediments. The results indicate that all studied sediments harbored viable and coexisting nitrate- and sulfate-reducing communities, which were able to instantaneously consume the electron acceptors supplied to the reactors. On average, NRR exceeded SRR by about one order of magnitude (309±180nmol NO3- cm−3h−1versus 37±29nmolSO42- cm−3h−1). The NRR:SRR molar ratio, however, varied significantly from site to site, with values ranging from 1.7 to 59. Nitrite production, indicative of incomplete nitrate reduction, was observed in all studied sediments and, on average, accounted for 45% of NRR (range 3–80%). Production of sulfate under nitrate-reducing conditions was observed in 10 out of 14 of the studied sediments, suggesting a common occurrence of sulfide oxidation coupled to nitrate reduction. Oxidation of sulfide accounted for 0 to 40% of NRR in the nitrate-only experiments. When both electron acceptors were supplied simultaneously, net sulfate consumption decreased on average by 45%. The effect of nitrate on SRR was highly variable, however, ranging from near complete inhibition to a 25% enhancement of SRR. Overall, the results of this study point to the need to critically reassess the model formulations used to represent anaerobic respiration processes and their interactions in early diagenetic models.

Enhanced anoxic bioremediation of PAHs-contaminated sediment

In this study, the biodegradation of 16 polycyclic aromatic hydrocarbons (PAHs) in marine sediment was investigated under three different anoxic conditions, i.e. sulfate-only, nitrate-only and mixed nitrate/sulfate as electron acceptors. All two-, three- and four-ring PAHs showed significant biodegradation with the removal efficiencies ranging from 42% to 77%, while five- and six-ring PAHs showed little degradation. The results illustrated that two- to three-ring PAHs could be degraded at a rate of 4.01×10−2–6.42×10−2d−1 under nitrate-reducing condition, faster than that of under sulfate-reducing condition. Biodegradation of two- and three-ring PAHs followed first-order model well with the rate constants of 1.62×10−2–6.42×10−2d−1. The biodegradation of four ring PAHs followed the zero-order kinetic model with the rate constants of 1.26×10−2–2.22×10−2mg/kg/d. Molecular analysis indicated that nahAc gene increased by two orders of magnitude during the biodegradation and served as a good indicator of PAHs-degrading bacterial population and biodegradation process.

Changes in iso- and n-alkane distribution during biodegradation of crude oil under nitrate and sulphate reducing conditions

Crude oil consists of a large number of hydrocarbons with different susceptibility to microbial degradation. The influence of hydrocarbon structure and molecular weight on hydrocarbon biodegradation under anaerobic conditions is not fully explored. In this study oxygen, nitrate and sulphate served as terminal electron acceptors (TEAs) for the microbial degradation of a paraffin-rich crude oil in a freshly contaminated soil. During 185 days of incubation, alkanes from n-C11 to n-C39, three n- to iso-alkane ratios commonly used as weathering indicators and the unresolved complex mixture (UCM) were quantified and statistically analyzed. The use of different TEAs for hydrocarbon degradation resulted in dissimilar degradative patterns for n- and iso-alkanes. While n-alkane biodegradation followed well-established patterns under aerobic conditions, lower molecular weight alkanes were found to be more recalcitrant than mid- to high-molecular weight alkanes under nitrate-reducing conditions. Biodegradation with sulphate as the TEA was most pronounced for long-chain (n-C32 to n-C39) alkanes. The observation of increasing ratios of n-C17 to pristane and of n-C18 to phytane provides first evidence of the preferential degradation of branched over normal alkanes under sulphate reducing conditions. The formation of distinctly different n- and iso-alkane biodegradation fingerprints under different electron accepting conditions may be used to assess the occurrence of specific degradation processes at a contaminated site. The use of n- to iso-alkane ratios for this purpose may require adjustment if applied for anaerobic sites.

Anaerobic biodegradation of ethylthionocarbamate by the mixed bacteria under various electron acceptor conditions

Biodegradation behavior and kinetics of ethylthionocarbamate under nitrate, sulfate and ferric reducing conditions by mixed cultures enriched from the anaerobic digester sludge was investigated. The results showed that ethylthionocarbamate could be degraded independently by the mixed cultures coupled to nitrate, sulfate, and ferric reduction, and meanwhile, nitrite, sulfide, and ferrous were accumulated as a result of nitrate, sulfate and ferric reduction, respectively. Ferric was a more favorable terminal electron acceptor compared to nitrate and sulfate. The order of the electron acceptors with decreasing biodegradation rates of the ethylthionocarbamate was: ferric>nitrate>sulfate, and the corresponding maximum biodegradation rate was 7.240, 6.267, and 4.602mg/(L·d), respectively. The anaerobic biodegradation of ethylthionocarbamate under various electron acceptor conditions can be accurately described by first order exponential decay kinetics.

Behaviour of PAHs during sewage sludge fermentation in the presence of sulphate and nitrate

In this study, the changes of polycyclic aromatic hydrocarbons (PAHs) in sewage sludge and supernatants during anaerobic digestion under nitrate- and sulphate-reducing conditions were investigated. NaNO 3 or Na 2 SO 4 were added to the mixed sludge in order to obtain denitrification and sulphate reduction conditions, respectively. Abiotic loses of PAHs simultaneously were observed. Abiotic samples (with sodium azide) were also prepared. All sludge samples (biotic and abiotic) were incubated at 35°C in the dark. PAHs concentration was determined before incubation, after 13 d and after 20 d of incubation. Quantification of 16 PAHs (EPA) was carried out by GC-MS. PAHs were determined both in sewage sludge and in supernatants. During hydrolysis of organic compounds (first phase of fermentation) in the control samples (in sewage sludge and in superna tants) the increase of PAHs concentration occurred (especially naphthalene and 3-ring PAHs). The concentration of 16 PAHs in sewage sludge was reduced by 30% on average under nitrate-reducing conditions after 20 d and by 32% on average under sulphate-reducing conditions, respectively. The effectiveness of reducing of individual hydrocarbons was different. The concentration of carcino genic PAHs in sewage sludge was reduced by 73% on average under nitrate-reducing conditions and by 79% on average under sulphate-reducing conditions, respectively.

Geochemical and Microbial Controls of the Decomposition of Depleted Uranium in the Environment: Experimental Studies using Soil Microorganisms

The decomposition of depleted uranium (DU) alloy in the presence of microorganisms characteristic of clay-rich soils has been studied using microcosm experiments. To assess the possible roles of specific groups of soil microorganisms, enrichment culture experiments were undertaken where the indigenous microbes were isolated from the soil and selectively cultured by adding growth medium supplemented with a specific terminal electron acceptor and electron donor, producing an inoculum to which a DU “coupon” was added. Experiments were conducted using microcosms with enriched consortia of either aerobic or anaerobic bacteria, including fermentative organisms and those that respire Fe(III), sulfate or nitrate. In a series of experiments, the rate and extent of DU breakdown was determined for each of the consortia. Changes in solution chemistry (pH, Eh, total Fe, Fe(II), nitrate, sulfate, glucose) were monitored and the proportions of dissolved uranium and solid breakdown products determined. The latter were characterized using environmental scanning electron microscopy (ESEM), X-ray powder diffractometry (XRD), and X-ray photoelectron spectroscopy (XPS). The microbial communities in the microcosms exposed to the DU were studied using PCR-based 16SrRNA profiling techniques. In the aerobic microcosms, significant DU corrosion as determined by mass loss (of ∼1.8%) occurred over 40 days; however, within experimental error, as much or more DU was consumed (∼3% weight loss) in an abiotic control experiment. The slower rate in the biotic experiment may reflect formation of a passivating film at the surface. Under anaerobic conditions, DU corrosion was less extensive than under aerobic conditions, with abiotic control experiments again showing comparable or greater mass loss. The order of decreasing corrosion rates under abiotic anaerobic conditions was: Fe (III)-reducing conditions (∼3% mass loss) > fermenting conditions (∼1% mass loss) > nitrate-reducing conditions (∼0.2% mass loss) > sulfate-reducing conditions (∼0.1% mass loss). In the equivalent biotic systems, corrosion was only significantly different to the abiotic treatments under Fe(III)-reducing conditions (∼2% mass loss), fermenting conditions (∼0.1% mass loss) and under nitrate-reducing conditions where biotic corrosion was so slow that mass loss was only measurable after extending the experiment to 300 days (after which there was 0.2% mass loss). The microbial communities in the different enrichment cultures were distinct with no evidence of differences between the microbial communities sampled from the DU coupon surface and those from the ‘planktonic’ population. After 40 days of corrosion in the aerobic systems, pale yellow, black and bright yellow alteration products had formed. These products were identified as chernikovite, uraninite and schoepite, respectively. The limited extent of alteration in the anaerobic systems did not yield identifiable mineral alteration products. Overall, these experiments show that microbes are surprisingly ineffective at promoting the breakdown of DU, which is dominated by chemical corrosion and leads to end-products that reflect the geochemical environment in which the corrosion has occurred, and that may include relatively stable mineral phases. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental file.

Microaerophilic, Fe(II)-dependent growth and Fe(II) oxidation by a Dechlorospirillum species

A species of Dechlorospirillum was isolated from an Fe(II)-oxidizing, opposing-gradient-culture enrichment using an inoculum from a circumneutral, freshwater creek that showed copious amounts of Fe(III) (hydr)oxide precipitation. In gradient cultures amended with a redox indicator to visualize the depth of oxygen penetration, Dechlorospirillum sp. strain M1 showed Fe(II)-dependent growth at the oxic-anoxic interface and was unable to utilize sulfide as an alternate electron donor. The bacterium also grew with acetate as an electron donor under both microaerophilic and nitrate-reducing conditions, but was incapable of organotrophic Fe(III) reduction or nitrate-dependent Fe(II) oxidation. Although members of the genus Dechlorospirillum are primarily known as perchlorate and nitrate reducers, our results suggest that some species are members of the microbial communities involved in iron redox cycling at the oxic-anoxic transition zones in freshwater sediments.

The Enhancement of Naphthalene Degradation in Soil by Hydroxypropyl-β-Cyclodextrin

The enriched anaerobic naphthalene-adapted mixed bacteria were capable of biodegrading naphthalene efficiently and without a lag phase in soil upon inoculating under nitrate reducing conditions when the initial concentration was below 150mg/kg in soil. Nitrite, a product of nitrate reduction, did not inhibit naphthalene degradation even at concentrations over 143mg/L. Naphthalene degradation was enhanced approximately two times when 10g/L hydroxypropyl-β-cyclodextrin (HPCD) was amended simultaneously in soil.

Biotransformation of 17α-methyltestosterone in sediment under different electron acceptor conditions

17α-Methyltestosterone (MT), an anabolic androgenic steroid, is used widely in inducing an all male population in aquaculture farming of fish, such as Nile tilapia ( Oreochromis niloticus). Current understanding of the occurrence and fate of MT in the sediments and the surrounding areas of the aquaculture ponds are very limited. Bioassay tests showed that MT was biotransformed under aerobic and sulfate-reducing conditions with a half-life of 3.8 d and 5.3 d, respectively, with complete disappearance of androgenic activity. However, under methanogenic condition, MT was found to biotransform but the androgenic activity continued to persist even after 45 d of incubation. In contrast, MT was found to transform slowly under iron(III)-reducing condition and was hardly transformed under nitrate-reducing condition. A possible reason for the lack of transformation of MT under nitrate-reducing condition is the presence of the methyl group at the C-17 position. The results of this study suggest that MT and its degradation products with androgenic activity may potentially accumulate in the sediments of fish farming ponds under iron(III)-reducing, nitrate-reducing and methanogenic conditions.

Preferential biodegradation of long chain hydrocarbons (C15-C29) under sulphate and nitrate reducing conditions

Preferential biodegradation of long chain hydrocarbons (C15-C29) under sulphate and nitrate reducing conditions

Optimization of anaerobic PAH degradation under nitrate reducing conditions in pilot scale experiments

Optimization of anaerobic PAH degradation under nitrate reducing conditions in pilot scale experiments

Relation between the activity of anaerobic microbial populations in oil sands tailings ponds and the sedimentation of tailings

Oil sands tailings ponds contain a variety of anaerobic microbes, including methanogens, sulfate- and nitrate-reducing bacteria. Methanogenic activity in samples from a tailings pond and its input streams was higher with trimethylamine (TMA) than with acetate. Methanogens closely affiliated to Methanomethylovorans hollandica were found in the TMA enrichments. Tailings sedimentation increased with methanogenic activity, irrespective whether TMA or acetate was used to stimulate methanogenesis. Increased sedimentation of autoclaved tailings was observed with added pure cultures under methanogenic, as well as under nitrate-reducing conditions, but not under sulfate-reducing conditions. Scanning electron microscopy and energy-dispersive X-ray spectroscopy indicated the presence of microbes and of extracellular polymeric substances in tailings particle aggregates, especially under methanogenic and nitrate-reducing conditions. Hence different classes of microorganisms growing in tailings ponds contribute to increased tailings aggregation and sedimentation. Because addition of nitrate is known to lower methane production by methanogenic consortia, these observations offer the potential to combine lower methane emissions with improved microbially-induced tailings sedimentation.

Isolation, genetic and functional characterization of novel soil nirK-type denitrifiers

Denitrification, the reduction of nitrogen oxides (NO 3 − and NO 2 −) to N 2 via the intermediates NO and N 2O, is crucial for nitrogen turnover in soils. Cultivation-independent approaches that applied nitrite reductase genes ( nirK/ nirS) as marker genes to detect denitrifiers showed a predominance of genes presumably derived from as yet uncultured organisms. However, the phylogenetic affiliation of these organisms remains unresolved since the ability to denitrify is widespread among phylogenetically unrelated organisms. In this study, denitrifiers were cultured using a strategy to generally enrich soil microorganisms. Of 490 colonies screened, eight nirK-containing isolates were phylogenetically identified (16S rRNA genes) as members of the Rhizobiales. A nirK gene related to a large cluster of sequences from uncultured bacteria mainly retrieved from soil was found in three isolates classified as Bradyrhizobium sp. Additional isolates were classified as Bradyrhizobium japonicum and Bosea sp. that contained nirK genes also closely related to the nirK from these strains. These isolates denitrified, albeit with different efficiencies. In Devosia sp., nirK was the only denitrification gene detected. Two Mesorhizobium sp. isolates contained a nirK gene also related to nirK from cultured Mesorhizobia and uncultured soil bacteria but no gene encoding nitric oxide or nitrous oxide reductase. These isolates accumulated NO under nitrate-reducing conditions without growth, presumably due to the lethal effects of NO. This showed the presence of a functional nitrite reductase but lack of a nitric oxide reductase. In summary, similar nirK genotypes recurrently detected mainly in soils likely originated from Rhizobia, and functional differences were presumably strain-dependent.

Degradation of 4-n-nonylphenol under nitrate reducing conditions

Nonylphenol (NP) is an endocrine disruptor present as a pollutant in river sediment. Biodegradation of NP can reduce its toxicological risk. As sediments are mainly anaerobic, degradation of linear (4-n-NP) and branched nonylphenol (tNP) was studied under methanogenic, sulphate reducing and denitrifying conditions in NP polluted river sediment. Anaerobic bioconversion was observed only for linear NP under denitrifying conditions. The microbial population involved herein was further studied by enrichment and molecular characterization. The largest change in diversity was observed between the enrichments of the third and fourth generation, and further enrichment did not affect the diversity. This implies that different microorganisms are involved in the degradation of 4-n-NP in the sediment. The major degrading bacteria were most closely related to denitrifying hexadecane degraders and linear alkyl benzene sulphonate (LAS) degraders. The molecular structures of alkanes and LAS are similar to the linear chain of 4-n-NP, this might indicate that the biodegradation of linear NP under denitrifying conditions starts at the nonyl chain. Initiation of anaerobic NP degradation was further tested using phenol as a structure analogue. Phenol was chosen instead of an aliphatic analogue, because phenol is the common structure present in all NP isomers while the structure of the aliphatic chain differs per isomer. Phenol was degraded in all cases, but did not affect the linear NP degradation under denitrifying conditions and did not initiate the degradation of tNP and linear NP under the other tested conditions.