scholarly journals Methanogenic Paraffin Biodegradation: Alkylsuccinate Synthase Gene Quantification and Dicarboxylic Acid Production

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Lisa K. Oberding ◽  
Lisa M. Gieg

ABSTRACTParaffinicn-alkanes (>C17) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffinn-octacosane (C28H58). Compared with that in controls, the consumption ofn-octacosane was coupled to methane production, demonstrating its biodegradation under these conditions.Smithellawas postulated to be an important C28H58degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identifiedassAgene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from otherSmithellaorganisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays forassAdemonstrated significant increases in the abundance and expression of this gene in C28H58-degrading cultures compared with that in controls, suggestingn-octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C28H58-degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery.IMPORTANCEUnderstanding the methanogenic biodegradation of different classes of hydrocarbons has important applications for effective fuel-contaminated site remediation and for improved recovery from oil reservoirs. Previous studies have clearly demonstrated that short-chain alkanes (<C17) can be biodegraded anaerobically, but less is understood regarding the biodegradation of longer chain waxy alkanes (>C17) that comprise many fuel mixtures. Using an enrichment culture derived from a freshwater fuel-contaminated site, we demonstrate that the model waxy alkanen-octacosane can be biodegraded under methanogenic conditions by a presumedSmithellaphylotype. Compared with that of controls, we show an increased abundance and expression of theassAgene, which is known to be important for anaerobicn-alkane metabolism. Metabolite analyses revealed the presence of a range of α,ω-dicarboxylic acids found only inn-octacosane-degrading cultures, a novel finding that lends insight as to how anaerobic communities may access waxes as growth substrates in anoxic environments.

2013 ◽  
Vol 79 (15) ◽  
pp. 4701-4711 ◽  
Author(s):  
Richard Villemur ◽  
Silvia Cristina Cunha dos Santos ◽  
Julianne Ouellette ◽  
Pierre Juteau ◽  
François Lépine ◽  
...  

ABSTRACTNaturally occurring and synthetic estrogens and other molecules from industrial sources strongly contribute to the endocrine disruption of urban wastewater. Because of the presence of these molecules in low but effective concentrations in wastewaters, these endocrine disruptors (EDs) are only partially removed after most wastewater treatments, reflecting the presence of these molecules in rivers in urban areas. The development of a two-phase partitioning bioreactor (TPPB) might be an effective strategy for the removal of EDs from wastewater plant effluents. Here, we describe the establishment of three ED-degrading microbial enrichment cultures adapted to a solid-liquid two-phase partitioning system using Hytrel as the immiscible water phase and loaded with estrone, estradiol, estriol, ethynylestradiol, nonylphenol, and bisphenol A. All molecules except ethynylestradiol were degraded in the enrichment cultures. The bacterial composition of the three enrichment cultures was determined using 16S rRNA gene sequencing and showed sequences affiliated with bacteria associated with the degradation of these compounds, such asSphingomonadales. OneRhodococcusisolate capable of degrading estrone, estradiol, and estriol was isolated from one enrichment culture. These results highlight the great potential for the development of TPPB for the degradation of highly diluted EDs in water effluents.


2013 ◽  
Vol 80 (3) ◽  
pp. 1126-1131 ◽  
Author(s):  
Taiki Katayama ◽  
Manabu Kanno ◽  
Naoki Morita ◽  
Tomoyuki Hori ◽  
Takashi Narihiro ◽  
...  

ABSTRACTMedium- and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the familyErysipelotrichaceaein the phylumFirmicutesbut is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited β-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.


Author(s):  
Sinchan Banerjee ◽  
András Táncsics ◽  
Erika Tóth ◽  
Fruzsina Révész ◽  
Károly Bóka ◽  
...  

A benzene, para- and meta-xylene-degrading Gram-stain-negative, aerobic, yellow-pigmented bacterium, designated as D2P1T, was isolated from a para-xylene-degrading enrichment culture. Phylogenetic analyses based on 16S rRNA genes showed that D2P1T shares a distinct phyletic lineage within the genus Hydrogenophaga and shows highest 16S rRNA gene sequence similarity to Hydrogenophaga taeniospiralis NBRC 102512T (99.2 %) and Hydrogenophaga palleronii NBRC 102513T (98.3 %). The draft genome sequence of D2P1T is 5.63 Mb long and the genomic DNA G+C content is 65.5 %. Orthologous average nucleotide identity (OrthoANI) and digital DNA–DNA hybridization (dDDH) analyses confirmed low genomic relatedness to its closest relatives (OrthoANI <86 %; dDDH <30 %). D2P1T contains ubiquinone 8 (Q-8) as the only respiratory quinone and phospholipid, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol as major polar lipids. The main whole-cell fatty acids of D2P1T are summed feature 3 (C16 : 1  ω7c/C16 : 1  ω6c), C16 : 0 and summed feature 8 (C18 : 1  ω7c/C18 : 1  ω6c). The polyphasic taxonomic results indicated that strain D2P1T represents a novel species of the genus Hydrogenophaga , for which the name Hydrogenophaga aromaticivorans sp. nov. is proposed. The type strain is D2P1T (=LMG 31780T=NCAIM B 02655T).


2018 ◽  
Vol 84 (9) ◽  
Author(s):  
Claudia Tominski ◽  
Helene Heyer ◽  
Tina Lösekann-Behrens ◽  
Sebastian Behrens ◽  
Andreas Kappler

ABSTRACTMost isolated nitrate-reducing Fe(II)-oxidizing microorganisms are mixotrophic, meaning that Fe(II) is chemically oxidized by nitrite that forms during heterotrophic denitrification, and it is debated to which extent Fe(II) is enzymatically oxidized. One exception is the chemolithoautotrophic enrichment culture KS, a consortium consisting of a dominant Fe(II) oxidizer,Gallionellaceaesp., and less abundant heterotrophic strains (e.g.,Bradyrhizobiumsp.,Nocardioidessp.). Currently, this is the only nitrate-reducing Fe(II)-oxidizing culture for which autotrophic growth has been demonstrated convincingly for many transfers over more than 2 decades. We used 16S rRNA gene amplicon sequencing and physiological growth experiments to analyze the community composition and dynamics of culture KS with various electron donors and acceptors. Under autotrophic conditions, an operational taxonomic unit (OTU) related to known microaerophilic Fe(II) oxidizers within the familyGallionellaceaedominated culture KS. With acetate as an electron donor, most 16S rRNA gene sequences were affiliated withBradyrhizobiumsp.Gallionellaceaesp. not only was able to oxidize Fe(II) under autotrophic and mixotrophic conditions but also survived over several transfers of the culture on only acetate, although it then lost the ability to oxidize Fe(II).Bradyrhizobiumspp. became and remained dominant when culture KS was cultivated for only one transfer under heterotrophic conditions, even when conditions were reverted back to autotrophic in the next transfer. This study showed a dynamic microbial community in culture KS that responded to changing substrate conditions, opening up questions regarding carbon cross-feeding, metabolic flexibility of the individual strains in KS, and the mechanism of Fe(II) oxidation by a microaerophile in the absence of O2.IMPORTANCENitrate-reducing Fe(II)-oxidizing microorganisms are present in aquifers, soils, and marine and freshwater sediments. Most nitrate-reducing Fe(II) oxidizers known are mixotrophic, meaning that they need organic carbon to continuously oxidize Fe(II) and grow. In these microbes, Fe(II) was suggested to be chemically oxidized by nitrite that forms during heterotrophic denitrification, and it remains unclear whether or to what extent Fe(II) is enzymatically oxidized. In contrast, the enrichment culture KS was shown to oxidize Fe(II) autotrophically coupled to nitrate reduction. This culture contains the designated Fe(II) oxidizerGallionellaceaesp. and several heterotrophic strains (e.g.,Bradyrhizobiumsp.). We showed that culture KS is able to metabolize Fe(II) and a variety of organic substrates and is able to adapt to dynamic environmental conditions. When the community composition changed andBradyrhizobiumbecame the dominant community member, Fe(II) was still oxidized byGallionellaceaesp., even when culture KS was cultivated with acetate/nitrate [Fe(II) free] before being switched back to Fe(II)/nitrate.


2006 ◽  
Vol 73 (4) ◽  
pp. 1332-1340 ◽  
Author(s):  
Masashi Hatamoto ◽  
Hiroyuki Imachi ◽  
Akiyoshi Ohashi ◽  
Hideki Harada

ABSTRACT We investigated long-chain fatty acid (LCFA)-degrading anaerobic microbes by enrichment, isolation, and RNA-based stable isotope probing (SIP). Primary enrichment cultures were made with each of four LCFA substrates (palmitate, stearate, oleate, or linoleate, as the sole energy source) at 55�C or 37�C with two sources of anaerobic granular sludge as the inoculum. After several transfers, we obtained seven stable enrichment cultures in which LCFAs were converted to methane. The bacterial populations in these cultures were then subjected to 16S rRNA gene-based cloning, in situ hybridization, and RNA-SIP. In five of seven enrichment cultures, the predominant bacteria were affiliated with the family Syntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylum Firmicutes and the class Deltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the family Syntrophomonadaceae, and strain TOL, affiliated with the phylum Firmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association with Methanospirillum hungatei. Even though strain TOL assimilated [13C]palmitate in the original enrichment culture, strain TOL has not shown the ability to degrade LCFAs after isolation. These results suggest that microbes involved in the degradation of LCFAs under methanogenic conditions might not belong only to the family Syntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.


2011 ◽  
Vol 77 (17) ◽  
pp. 6100-6108 ◽  
Author(s):  
Dae-Wi Kim ◽  
Thomas M. Heinze ◽  
Bong-Soo Kim ◽  
Laura K. Schnackenberg ◽  
Kellie A. Woodling ◽  
...  

ABSTRACTAntimicrobial residues found in municipal wastewater may increase selective pressure on microorganisms for development of resistance, but studies with mixed microbial cultures derived from wastewater have suggested that some bacteria are able to inactivate fluoroquinolones. Medium containingN-phenylpiperazine and inoculated with wastewater was used to enrich fluoroquinolone-modifying bacteria. One bacterial strain isolated from an enrichment culture was identified by 16S rRNA gene sequence analysis as aMicrobacteriumsp. similar to a plant growth-promoting bacterium,Microbacterium azadirachtae(99.70%), and a nematode pathogen, “M. nematophilum” (99.02%). During growth in medium with norfloxacin, this strain produced four metabolites, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) analyses as 8-hydroxynorfloxacin, 6-defluoro-6-hydroxynorfloxacin, desethylene norfloxacin, andN-acetylnorfloxacin. The production of the first three metabolites was enhanced by ascorbic acid and nitrate, but it was inhibited by phosphate, amino acids, mannitol, formate, and thiourea. In contrast,N-acetylnorfloxacin was most abundant in cultures supplemented with amino acids. This is the first report of defluorination and hydroxylation of a fluoroquinolone by an isolated bacterial strain. The results suggest that some bacteria may degrade fluoroquinolones in wastewater to metabolites with less antibacterial activity that could be subject to further degradation by other microorganisms.


2012 ◽  
Vol 194 (18) ◽  
pp. 5147-5148 ◽  
Author(s):  
Steven D. Brown ◽  
Mircea Podar ◽  
Dawn M. Klingeman ◽  
Courtney M. Johnson ◽  
Zamin K. Yang ◽  
...  

ABSTRACTPelosinus fermentans16S rRNA gene sequences have been reported from diverse geographical sites since the recent isolation of the type strain. We present the genome sequence of theP. fermentanstype strain R7 (DSM 17108) and genome sequences for two new strains with different abilities to reduce iron, chromate, and uranium.


2006 ◽  
Vol 73 (4) ◽  
pp. 1054-1064 ◽  
Author(s):  
Diana Z. Sousa ◽  
M. Alcina Pereira ◽  
Alfons J. M. Stams ◽  
M. Madalena Alves ◽  
Hauke Smidt

ABSTRACTAnaerobic long-chain fatty acid (LCFA)-degrading bacteria were identified by combining selective enrichment studies with molecular approaches. Two distinct enrichment cultures growing on unsaturated and saturated LCFAs were obtained by successive transfers in medium containing oleate and palmitate, respectively, as the sole carbon and energy sources. Changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in oleate and palmitate enrichment cultures clustered with fatty acid-oxidizing bacteria withinSyntrophomonadaceaeandSyntrophobacteraceaefamilies. A low methane yield, corresponding to 9 to 18% of the theoretical value, was observed in the oleate enrichment, and acetate, produced according to the expected stoichiometry, was not further converted to methane. In the palmitate enrichment culture, the acetate produced was completely mineralized and a methane yield of 48 to 70% was achieved from palmitate degradation. Furthermore, the oleate enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate-specialized consortia degraded oleate only after a lag phase of 3 months, after which the DGGE profile had changed. Two predominant bands appeared, and sequence analysis showed affiliation with theSyntrophomonasgenus. These bands were also present in the oleate enrichment culture, suggesting that these bacteria are directly involved in oleate degradation, emphasizing possible differences between the degradation of unsaturated and saturated LCFAs.


2013 ◽  
Vol 63 (Pt_7) ◽  
pp. 2410-2417 ◽  
Author(s):  
Shih-Yao Lin ◽  
Asif Hameed ◽  
You-Cheng Liu ◽  
Yi-Han Hsu ◽  
Wei-An Lai ◽  
...  

An aerobic, Gram-stain-negative, rod-shaped bacterium with a single polar flagellum, designated CC-OPY-1T, was isolated from an oil-contaminated site in Taiwan. CC-OPY-1T produces siderophores, and can grow at temperatures of 25–37 °C and pH 5.0–9.0 and tolerate <5 % (w/v) NaCl. The 16S rRNA gene sequence analysis of CC-OPY-1T showed high pairwise sequence similarity to Pseudomonas alcaligenes BCRC 11893T (97.1 %), Pseudomonas. alcaliphila DSM 17744T (97.1 %), Pseudomonas tuomuerensis JCM 14085T (97.1 %), Pseudomonas toyotomiensis JCM 15604T (96.9 %) and lower sequence similarity to remaining species of the genus Pseudomonas . The phylogenetic trees reconstructed based on gyrB and rpoB gene sequences supported the classification of CC-OPY-1T as a novel member of the genus Pseudomonas . The predominant quinone system of strain CC-OPY-1T was ubiquinone (Q-9) and the DNA G+C content was 68.4±0.3 mol%. The major fatty acids were C12 : 0, C16 : 0, C17 : 0 cyclo and summed features 3 and 8 consisting of C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c, respectively. The major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC) and two unknown phospholipids (PL1–2). Due to distinct phylogenetic, phenotypic and chemotaxonomic features, CC-OPY-1T is proposed to represent a novel species within the genus Pseudomonas for which the name Pseudomonas sagittaria sp. nov. is proposed. The type strain is CC-OPY-1T ( = BCRC 80399T = JCM 18195T).


2013 ◽  
Vol 63 (Pt_8) ◽  
pp. 3050-3055 ◽  
Author(s):  
K. V. N. S. Lakshmi ◽  
Ch. Sasikala ◽  
E. V. V. Ramaprasad ◽  
Ch. V. Ramana

A reddish-brown bacterium, designated strain JA318T, was purified from a photoheterotrophic enrichment culture obtained from the rhizosphere soil of paddy. Cells of strain JA318T are spiral shaped, Gram-stain-negative and motile by means of amphitrichous flagella. Strain JA318T has no NaCl requirement for growth but can tolerate up to 1.5 % (w/v) NaCl. Internal photosynthetic membranes are present as lamellar stacks. Photoorganoheterotrophy is the only growth mode observed. Strain JA318T contains bacteriochlorophyll a, lycopene and rhodopin as major carotenoids. Thiamine, niacin and para-aminobenzoic acid (PABA) are required as growth factors. Major fatty acids are C18 : 1ω7c and C16 : 0. Ubiquinone-8 and rhodoquinone-8 are the observed quinones. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid are the major polar lipids in strain JA318T. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain JA318T clustered with species of the genus Rhodospirillum which belongs to the class Alphaproteobacteria . The highest sequence similarity of strain JA318T was found with Rhodospirillum sulfurexigens JA143T (99.9 %). The DNA–DNA reassociation values of strain JA318T with Rsp. sulfurexigens JA143T and Rhodospirillum photometricum DSM 122T were 52±2 % and 45±1 %, respectively. The genomic DNA G+C content of strain JA318T was 60.2 mol%. Based on the morphological, physiological, chemotaxonomical and molecular evidence, strain JA318T is significantly different from the type strains of species of the genus Rhodospirillum , of the family Rhodospirillaceae , and it is proposed that the strain be classified as a representative of a novel species for which the name Rhodospirillum oryzae sp. nov. is proposed. The type strain is JA318T ( = KCTC 5960T = NBRC 107573T).


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