Eight Metagenome-Assembled Genomes Provide Evidence for Microbial Adaptation in 20,000- to 1,000,000-Year-Old Siberian Permafrost.

Permafrost microbes may be metabolically active in microscopic layers of liquid brines, even in ancient soil. Metagenomics can help discern whether permafrost microbes show adaptations to this environment. Thirty-three metagenome-assembled genomes (MAGs) were obtained from six depths (3.5 m to 20 m) of freshly cored permafrost from the Siberian Kolyma-Indigirka Lowland region. These soils have been continuously frozen for ∼20,000 to 1,000,000 years. Eight of these MAGs were ≥80% complete with <10% contamination and were taxonomically identified as Aminicenantes, Atribacteria, Chloroflexi, and Actinobacteria within bacteria and Thermoprofundales within archaea. MAGs from these taxa have been obtained previously from nonpermafrost environments and have been suggested to show adaptations to long-term energy starvation, but they have never been explored in ancient permafrost. The permafrost MAGs had greater proportions in the Clusters of Orthologous Groups (COGs) categories of energy production and conversion and carbohydrate transport and metabolism than did their nonpermafrost counterparts. They also contained genes for trehalose synthesis, thymine metabolism, mevalonate biosynthesis, and cellulose degradation, which were less prevalent in nonpermafrost genomes. Many of these genes are involved in membrane stabilization and osmotic stress responses, consistent with adaptation to the anoxic, high-ionic-strength, cold environments of permafrost brine films. Our results suggest that this ancient permafrost contains DNA of high enough quality to assemble MAGs from microorganisms with adaptations to survive long-term freezing in this extreme environment. IMPORTANCE Permafrost around the world is thawing rapidly. Many scientists from a variety of disciplines have shown the importance of understanding what will happen to our ecosystem, commerce, and climate when permafrost thaws. The fate of permafrost microorganisms is connected to these predicted rapid environmental changes. Studying ancient permafrost with culture-independent techniques can give a glimpse into how these microorganisms function under these extreme low-temperature and low-energy conditions. This will facilitate understanding how they will change with the environment. This study presents genomic data from this unique environment ∼20,000 to 1,000,000 years of age.

Aspartic acid racemization and repair in the survival and recovery of hyperthermophiles after prolonged starvation at high temperature.

Long-term survivability is well-known for microorganisms in nutrient-depleted environments, but the damage accrued by proteins and the associated repair processes during the starvation and recovery phase of microbial life still remain enigmatic. We focused on aspartic acid (Asp) racemization and repair in the survival of Pyrococcus furiosus and Thermococcus litoralis under starvation conditions at high temperature. Despite the dramatic decrease of viability over time, 0.002% of P. furiosus cells (2.1×103 cells/mL) and 0.23% of T. litoralis cells (2.3×105 cells/mL) remained viable after 25 and 50 days, respectively. The D/L Asp ratio in the starved cells was approximately half of those from the autoclaved cells, suggesting that the starving cells were capable of partially repairing racemized Asp. Transcriptomic analyses of the recovered cells of T. litoralis indicated that the gene encoding Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) might be involved in the repair of damaged proteins by converting D-Asp back to L-Asp during the resuscitation of starved cells. Collectively, our results provided evidence that Asp underwent racemization in the surviving hyperthermophilic cells under starved conditions and PIMT played a critical role in the repair of abnormal aspartyl residues during the initial recovery of starved, yet still viable, cells.

Genomic reconstruction of fossil and living microorganisms in ancient Siberian permafrost.

BACKGROUND: Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination between fossil and living microorganisms in ancient cryogenic environments. DNA repair protocols were combined with high throughput sequencing (HTS) of separate iDNA and eDNA fraction to reconstruct metagenome-assembled genomes (MAGs) from ancient microbial DNA entrapped in Siberian coastal permafrost. RESULTS: Despite the severe DNA damage in ancient permafrost, the coupling of DNA repair and HTS resulted in a total of 52 MAGs from sediments across a chronosequence (26-120 kyr). These MAGs were compared with those derived from the same samples but without utilizing DNA repair protocols. The MAGs from the youngest stratum showed minimal DNA damage and thus likely originated from viable, active microbial species. Many MAGs from the older and deeper sediment appear related to past aerobic microbial populations that had died upon freezing. MAGs from anaerobic lineages, including Asgard archaea, however exhibited minimal DNA damage and likely represent extant living microorganisms that have become adapted to the cryogenic and anoxic environments. The integration of aspartic acid racemization modeling and metaproteomics further constrained the metabolic status of the living microbial populations. Collectively, combining DNA repair protocols with HTS unveiled the adaptive strategies of microbes to long-term survivability in ancient permafrost. CONCLUSIONS: Our results indicated that coupling of DNA repair protocols with simultaneous sequencing of iDNA and eDNA fractions enabled the assembly of MAGs from past and living microorganisms in ancient permafrost. The genomic reconstruction from the past and extant microbial populations expanded our understanding about the microbial successions and biogeochemical alterations from the past paleoenvironment to the present-day frozen state. Furthermore, we provided genomic insights into long-term survival mechanisms of microorganisms under cryogenic conditions through geological time. The combined strategies in this study can be extrapolated to examine other ancient non-permafrost environments and constrain the search for past and extant extraterrestrial life in permafrost and ice deposits on Mars. Video abstract.

Permafrost Active Layer Microbes From Ny Ålesund, Svalbard (79°N) Show Autotrophic and Heterotrophic Metabolisms With Diverse Carbon-Degrading Enzymes.

The active layer of permafrost in Ny Ålesund, Svalbard (79°N) around the Bayelva River in the Leirhaugen glacier moraine is measured as a small net carbon sink at the brink of becoming a carbon source. In many permafrost-dominating ecosystems, microbes in the active layers have been shown to drive organic matter degradation and greenhouse gas production, creating positive feedback on climate change. However, the microbial metabolisms linking the environmental geochemical processes and the populations that perform them have not been fully characterized. In this paper, we present geochemical, enzymatic, and isotopic data paired with 10 Pseudomonas sp. cultures and metagenomic libraries of two active layer soil cores (BPF1 and BPF2) from Ny Ålesund, Svalbard, (79°N). Relative to BPF1, BPF2 had statistically higher C/N ratios (15 ± 1 for BPF1 vs. 29 ± 10 for BPF2; n = 30, p < 10-5), statistically lower organic carbon (2% ± 0.6% for BPF1 vs. 1.6% ± 0.4% for BPF2, p < 0.02), statistically lower nitrogen (0.1% ± 0.03% for BPF1 vs. 0.07% ± 0.02% for BPF2, p < 10-6). The d13C values for inorganic carbon did not correlate with those of organic carbon in BPF2, suggesting lower heterotrophic respiration. An increase in the δ13C of inorganic carbon with depth either reflects an autotrophic signal or mixing between a heterotrophic source at the surface and a lithotrophic source at depth. Potential enzyme activity of xylosidase and N-acetyl-ß-D-glucosaminidase increases twofold at 15°C, relative to 25°C, indicating cold adaptation in the cultures and bulk soil. Potential enzyme activity of leucine aminopeptidase across soils and cultures was two orders of magnitude higher than other tested enzymes, implying that organisms use leucine as a nitrogen and carbon source in this nutrient-limited environment. Besides demonstrating large variability in carbon compositions of permafrost active layer soils only ∼84 m apart, results suggest that the Svalbard active layer microbes are often limited by organic carbon or nitrogen availability and have adaptations to the current environment, and metabolic flexibility to adapt to the warming climate.

Genome-centric resolution of novel microbial lineages in an excavated Centrosaurus dinosaur fossil bone from the Late Cretaceous of North America.

BACKGROUND: Exceptional preservation of endogenous organics such as collagens and blood vessels has been frequently reported in Mesozoic dinosaur fossils. The persistence of these soft tissues in Mesozoic fossil bones has been challenged because of the susceptibility of proteins to degradation and because bone porosity allows microorganisms to colonize the inner microenvironments through geological time. Although protein lability has been studied extensively, the genomic diversity of microbiomes in dinosaur fossil bones and their potential roles in bone taphonomy remain underexplored. Genome-resolved metagenomics was performed, therefore, on the microbiomes recovered from a Late Cretaceous Centrosaurus bone and its encompassing mudstone in order to provide insight into the genomic potential for microbial alteration of fossil bone. RESULTS: Co-assembly and binning of metagenomic reads resulted in a total of 46 high-quality metagenome-assembled genomes (MAGs) affiliated to six bacterial phyla (Actinobacteria, Proteobacteria, Nitrospira, Acidobacteria, Gemmatimonadetes and Chloroflexi) and 1 archaeal phylum (Thaumarchaeota). The majority of the MAGs represented uncultivated, novel microbial lineages from class to species levels based on phylogenetics, phylogenomics and average amino acid identity. Several MAGs from the classes Nitriliruptoria, Deltaproteobacteria and Betaproteobacteria were highly enriched in the bone relative to the adjacent mudstone. Annotation of the MAGs revealed that the distinct putative metabolic functions of different taxonomic groups were linked to carbon, nitrogen, sulfur and iron metabolism. Metaproteomics revealed gene expression from many of the MAGs, but no endogenous collagen peptides were identified in the bone that could have been derived from the dinosaur. Estimated in situ replication rates among the bacterial MAGs suggested that most of the microbial populations in the bone might have been actively growing but at a slow rate. CONCLUSIONS: Our results indicate that excavated dinosaur bones are habitats for microorganisms including novel microbial lineages. The distinctive microhabitats and geochemistry of fossil bone interiors compared to that of the external sediment enrich a microbial biomass comprised of various novel taxa that harbor multiple gene sets related to interconnected biogeochemical processes. Therefore, the presence of these microbiomes in Mesozoic dinosaur fossils urges extra caution to be taken in the science of paleontology when hunting for endogenous biomolecules preserved from deep time.

Aspartic acid racemization constrains long-term viability and longevity of endospores.

Certain microorganisms survive long periods of time as endospores to cope with adverse conditions. Since endospores are metabolically inactive, the extent of aspartic acid (Asp) racemization will increase over time and might kill the spores by preventing their germination. Therefore, understanding the relationship between endospore survivability and Asp racemization is important for constraining the long-term survivability and global dispersion of spore-forming bacteria in nature. Geobacillus stearothermophilus was selected as a model organism to investigate racemization kinetics and survivability of its endospores at 65°C, 75°C and 98°C. This study found that the Asp racemization rates of spores and autoclaved spores were similar at all temperatures. The Asp racemization rate of spores was not significantly different from that of vegetative cells at 65°C. The Asp racemization rate of G. stearothermophilus spores was not significantly different from that of Bacillus subtilis spores at 98°C. The viability of spores and vegetative cells decreased dramatically over time, and the mortality of spores correlated exponentially with the degree of racemization (R2 = 0.9). This latter correlation predicts spore half-lives on the order of hundreds of years for temperatures typical of shallow marine sediments, a result consistent with studies about the survivability of thermophilic spores found in these environments.

Community succession in an anaerobic long-chain paraffin-degrading consortium and impact on chemical and electrical microbially influenced iron corrosion.

Community compositional changes and the corrosion of carbon steel in the presence of different electron donor and acceptor combinations were examined with a methanogenic consortium enriched for its ability to mineralize paraffins. Despite cultivation in the absence of sulfate, metagenomic analysis revealed the persistence of several sulfate-reducing bacterial taxa. Upon sulfate amendment, the consortium was able to couple C28H58 biodegradation with sulfate reduction. Comparative analysis suggested that Desulforhabdus and/or Desulfovibrio likely supplanted methanogens as syntrophic partners needed for C28H58 mineralization. Further enrichment in the absence of a paraffin revealed that the consortium could also utilize carbon steel as a source of electrons. The severity of both general and localized corrosion increased in the presence of sulfate, regardless of the electron donor utilized. With carbon steel as an electron donor, Desulfobulbus dominated in the consortium and electrons from iron accounted for ∼92% of that required for sulfate reduction. An isolated Desulfovibrio spp. was able to extract electrons from iron and accelerate corrosion. Thus, hydrogenotrophic partner microorganisms required for syntrophic paraffin metabolism can be readily substituted depending on the availability of an external electron acceptor and a single paraffin-degrading consortium harbored microbes capable of both chemical and electrical microbially influenced iron corrosion.

Predominance of Anaerobic, Spore-Forming Bacteria in Metabolically Active Microbial Communities from Ancient Siberian Permafrost.

The prevalence of microbial life in permafrost up to several million years (Ma) old has been well documented. However, the long-term survivability, evolution, and metabolic activity of the entombed microbes over this time span remain underexplored. We integrated aspartic acid (Asp) racemization assays with metagenomic sequencing to characterize the microbial activity, phylogenetic diversity, and metabolic functions of indigenous microbial communities across a ∼0.01- to 1.1-Ma chronosequence of continuously frozen permafrost from northeastern Siberia. Although Asp in the older bulk sediments (0.8 to 1.1 Ma) underwent severe racemization relative to that in the youngest sediment (∼0.01 Ma), the much lower d-Asp/l-Asp ratio (0.05 to 0.14) in the separated cells from all samples suggested that indigenous microbial communities were viable and metabolically active in ancient permafrost up to 1.1 Ma. The microbial community in the youngest sediment was the most diverse and was dominated by the phyla Actinobacteria and Proteobacteria In contrast, microbial diversity decreased dramatically in the older sediments, and anaerobic, spore-forming bacteria within Firmicutes became overwhelmingly dominant. In addition to the enrichment of sporulation-related genes, functional genes involved in anaerobic metabolic pathways such as fermentation, sulfate reduction, and methanogenesis were more abundant in the older sediments. Taken together, the predominance of spore-forming bacteria and associated anaerobic metabolism in the older sediments suggest that a subset of the original indigenous microbial community entrapped in the permafrost survived burial over geological time.IMPORTANCE Understanding the long-term survivability and associated metabolic traits of microorganisms in ancient permafrost frozen millions of years ago provides a unique window into the burial and preservation processes experienced in general by subsurface microorganisms in sedimentary deposits because of permafrost's hydrological isolation and exceptional DNA preservation. We employed aspartic acid racemization modeling and metagenomics to determine which microbial communities were metabolically active in the 1.1-Ma permafrost from northeastern Siberia. The simultaneous sequencing of extracellular and intracellular genomic DNA provided insight into the metabolic potential distinguishing extinct from extant microorganisms under frozen conditions over this time interval. This in-depth metagenomic sequencing advances our understanding of the microbial diversity and metabolic functions of extant microbiomes from early Pleistocene permafrost. Therefore, these findings extend our knowledge of the survivability of microbes in permafrost from 33,000 years to 1.1 Ma.

Cretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities.

Fossils were thought to lack original organic molecules, but chemical analyses show that some can survive. Dinosaur bone has been proposed to preserve collagen, osteocytes, and blood vessels. However, proteins and labile lipids are diagenetically unstable, and bone is a porous open system, allowing microbial/molecular flux. These 'soft tissues' have been reinterpreted as biofilms. Organic preservation versus contamination of dinosaur bone was examined by freshly excavating, with aseptic protocols, fossils and sedimentary matrix, and chemically/biologically analyzing them. Fossil 'soft tissues' differed from collagen chemically and structurally; while degradation would be expected, the patterns observed did not support this. 16S rRNA amplicon sequencing revealed that dinosaur bone hosted an abundant microbial community different from lesser abundant communities of surrounding sediment. Subsurface dinosaur bone is a relatively fertile habitat, attracting microbes that likely utilize inorganic nutrients and complicate identification of original organic material. There exists potential post-burial taphonomic roles for subsurface microorganisms.

The chances of establishing a real-world Jurassic Park are slim. During the fossilization process, biological tissues degrade over millions of years, with some types of molecules breaking down faster than others. However, traces of biological material have been found inside some fossils. While some researchers believe these could be the remains of ancient proteins, blood vessels, and cells, traditionally thought to be among the least stable components of bone, others think that they have more recent sources. One hypothesis is that they are in fact biofilms formed by bacteria. To investigate the source of the biological material in fossil bone, Saitta et al. performed a range of analyses on the fossilized bones of a horned dinosaur called Centrosaurus. The bones were carefully excavated in a manner to reduce contamination, and the sediment the bones had been embedded in was also tested for comparison. Saitta et al. found no evidence of ancient dinosaur proteins. However, the fossils contained more organic carbon, DNA, and certain amino acids than the sediment surrounding them. Most of these appeared to have a very recent source. Sequencing the genetic material revealed that the fossil had become a habitat for an unusual community of microbes that is not found in the surrounding sediment or above ground. These buried microbes may have evolved unique ways to thrive inside fossils. Future work could investigate how these unusual organisms live and whether the communities vary in different parts of the world.

Anaerobic biodegradation of biofuels and their impact on the corrosion of a Cu-Ni alloy in marine environments.

Fuel biodegradation linked to sulfate reduction can lead to corrosion of the metallic infrastructure in a variety of marine environments. However, the biological stability of emerging biofuels and their potential impact on copper-nickel alloys commonly used in marine systems has not been well documented. Two potential naval biofuels (Camelina-JP5 and Fisher-Tropsch-F76) and their petroleum-derived counterparts (JP5 and F76) were critically assessed in seawater/sediment incubations containing a metal coupon (70/30 Cu-Ni alloy). Relative to a fuel-unamended control (1.2 ±â€¯0.4 µM/d), Camelina-JP5 (86.4 ±â€¯1.6 µM/d) and JP5 (77.6 ±â€¯8.3 µM/d) stimulated much higher rates of sulfate reduction than either FT-F76 (11.4 ±â€¯2.7 µM/d) or F76 (38.4 ±â€¯3.7 µM/d). The general corrosion rate (r2 = 0.91) and pitting corrosion (r2 = 0.92) correlated with sulfate loss in these incubations. Despite differences in microbial community structure on the metal or in the aqueous or sediment phases, sulfate reducing bacteria affiliated with Desulfarculaceae and Desulfobacteraceae became predominant upon fuel amendment. The identification of alkylsuccinates and alkylbenzylsuccinates attested to anaerobic metabolism of fuel hydrocarbons. Sequences related to Desulfobulbaceae were highly enriched (34.2-64.8%) on the Cu-Ni metal surface, regardless of whether the incubation received a fuel amendment. These results demonstrate that the anaerobic metabolism of biofuel linked to sulfate reduction can exacerbate the corrosion of Cu-Ni alloys. Given the relative lability of Camelina-JP5, particular precaution should be taken when incorporating this hydroprocessed biofuel into marine environments serviced by a Cu-Ni metallic infrastructure.

Microbial activities in hydrocarbon-laden wastewaters: Impact on diesel fuel stability and the biocorrosion of carbon steel.

Anaerobic hydrocarbon biodegradation not only diminishes fuel quality, but also exacerbates the biocorrosion of the metallic infrastructure. While successional events in marine microbial ecosystems impacted by petroleum are well documented, far less is known about the response of communities chronically exposed to hydrocarbons. Shipboard oily wastewater was used to assess the biotransformation of different diesel fuels and their propensity to impact carbon steel corrosion. When amended with sulfate and an F76 military diesel fuel, the sulfate removal rate in the assay mixtures was elevated (26.8µM/d) relative to incubations receiving a hydroprocessed biofuel (16.1µM/d) or a fuel-unamended control (17.8µM/d). Microbial community analysis revealed the predominance of Anaerolineae and Deltaproteobacteria in F76-amended incubations, in contrast to the Beta- and Gammaproteobacteria in the original wastewater. The dominant Smithella-like sequences suggested the potential for syntrophic hydrocarbon metabolism. The general corrosion rate was relatively low (0.83 - 1.29±0.12mpy) and independent of the particular fuel, but pitting corrosion was more pronounced in F76-amended incubations. Desulfovibrionaceae constituted 50-77% of the sessile organisms on carbon steel coupons. Thus, chronically exposed microflora in oily wastewater were differentially acclimated to the syntrophic metabolism of traditional hydrocarbons but tended to resist isoalkane-laden biofuels.

Metabolic Capability of a Predominant Halanaerobium sp. in Hydraulically Fractured Gas Wells and Its Implication in Pipeline Corrosion.

Microbial activity associated with produced water from hydraulic fracturing operations can lead to gas souring and corrosion of carbon-steel equipment. We examined the microbial ecology of produced water and the prospective role of the prevalent microorganisms in corrosion in a gas production field in the Barnett Shale. The microbial community was mainly composed of halophilic, sulfidogenic bacteria within the order Halanaerobiales, which reflected the geochemical conditions of highly saline water containing sulfur species (S2O3 (2-), SO4 (2-), and HS(-)). A predominant, halophilic bacterium (strain DL-01) was subsequently isolated and identified as belonging to the genus Halanaerobium. The isolate could degrade guar gum, a polysaccharide polymer used in fracture fluids, to produce acetate and sulfide in a 10% NaCl medium at 37°C when thiosulfate was available. To mitigate potential deleterious effects of sulfide and acetate, a quaternary ammonium compound was found to be an efficient biocide in inhibiting the growth and metabolic activity of strain DL-01 relative to glutaraldehyde and tetrakis (hydroxymethyl) phosphonium sulfate. Collectively, our findings suggest that predominant halophiles associated with unconventional shale gas extraction could proliferate and produce sulfide and acetate from the metabolism of polysaccharides used in hydraulic fracturing fluids. These metabolic products might be returned to the surface and transported in pipelines to cause pitting corrosion in downstream infrastructure.

Anaerobic Biodegradation of Alternative Fuels and Associated Biocorrosion of Carbon Steel in Marine Environments.

Fuels that biodegrade too easily can exacerbate through-wall pitting corrosion of pipelines and tanks and result in unintentional environmental releases. We tested the biological stability of two emerging naval biofuels (camelina-JP5 and Fischer-Tropsch-F76) and their potential to exacerbate carbon steel corrosion in seawater incubations with and without a hydrocarbon-degrading sulfate-reducing bacterium. The inclusion of sediment or the positive control bacterium in the incubations stimulated a similar pattern of sulfate reduction with different inocula. However, the highest rates of sulfate reduction were found in incubations amended with camelina-JP5 [(57.2 ± 2.2)-(80.8 ± 8.1) µM/day] or its blend with petroleum-JP5 (76.7 ± 2.4 µM/day). The detection of a suite of metabolites only in the fuel-amended incubations confirmed that alkylated benzene hydrocarbons were metabolized via known anaerobic mechanisms. Most importantly, general (r(2) = 0.73) and pitting (r(2) = 0.69) corrosion were positively correlated with sulfate loss in the incubations. Thus, the anaerobic biodegradation of labile fuel components coupled with sulfate respiration greatly contributed to the biocorrosion of carbon steel. While all fuels were susceptible to anaerobic metabolism, special attention should be given to camelina-JP5 biofuel due to its relatively rapid biodegradation. We recommend that this biofuel be used with caution and that whenever possible extended storage periods should be avoided.

Roles of thermophilic thiosulfate-reducing bacteria and methanogenic archaea in the biocorrosion of oil pipelines.

Thermophilic sulfide-producing microorganisms from an oil pipeline network were enumerated with different sulfur oxyanions as electron acceptors at 55°C. Most-probable number (MPN) analysis showed that thiosulfate-reducing bacteria were the most numerous sulfidogenic microorganisms in pipeline inspection gauge (PIG) scrapings. Thiosulfate-reducing and methanogenic enrichments were obtained from the MPN cultures that were able to use yeast extract as the electron donor. Molecular analysis revealed that both enrichments harbored the same dominant bacterium, which belonged to the genus Anaerobaculum. The dominant archaeon in the methanogenic enrichment was affiliated with the genus Methanothermobacter. With yeast extract as the electron donor, the general corrosion rate by the thiosulfate-reducing enrichment (8.43 ± 1.40 milli-inch per year, abbreviated as mpy) was about 5.5 times greater than the abiotic control (1.49 ± 0.15 mpy), while the comparable measures for the methanogenic culture were 2.03 ± 0.49 mpy and 0.62 ± 0.07 mpy, respectively. Total iron analysis in the cultures largely accounted for the mass loss of iron measured in the weight loss determinations. Profilometry analysis of polished steel coupons incubated in the presence of the thiosulfate-reducing enrichment revealed 59 pits over an area of 71.16 mm(2), while only 6 pits were evident in the corresponding methanogenic incubations. The results show the importance of thiosulfate-utilizing, sulfide-producing fermentative bacteria such as Anaerobaculum sp. in the corrosion of carbon steel, but also suggest that Anaerobaculum sp. are of far less concern when growing syntrophically with methanogens.

Biodegradation of di-n-butyl phthalate by Rhodococcus sp. JDC-11 and molecular detection of 3, 4-phthalate dioxygenase gene.

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.

Degradation of di-n-butyl phthalate by newly isolated Ochrobactrum sp.

A gram negative isolate designated JDC-41 was obtained from river sludge using mixtures of phthalate esters as the sole source and energy. The isolate was identified as Ochrobactrum sp. based on its 16S rRNA gene sequence. Over 87% of supplied di-n-butyl phthalate (DBP) was degraded by JDC-41 in a pH neutral mineral salts medium at 30 degrees C within 48 h. Increased DBP (50-500 mg/L) in the culture correspondingly increased degradation half-life from 3.83 to 18.12 h. DBP induced cells more rapidly degraded DBP.

Genetic diversity of phthalic acid esters-degrading bacteria isolated from different geographical regions of China.

Thirty-two strains of phthalic acid ester (PAEs)-degrading bacteria were isolated from thirteen geographically diverse sites by enrichment using mixtures of PAEs as the sole source of carbon and energy. Sequence analyses of the 16S rRNA gene indicated that these isolates were from six genera (Arthrobacter, Gordonia, Rhodococcus, Acinetobacter, Pseudomonas, and Delftia). To evaluate the genetic diversity among them, the molecular typing method rep-PCR with primers based on enterobacterial repetitive intergenic consensus, repetitive extragenic palindromes, and BOXAIR sequences was performed. Strain-specific and unique genotypic fingerprints were distinguished for most of these isolates. In addition, utilization of various PAEs and the central intermediate phthalic acid by representative isolates suggested inter-isolate differences in the substrate utilization and degradation pathways. Furthermore, HPLC analysis showed that the rate of dimethyl phthalate degradation varied from 48.32 to 100% between strains. These results suggest a high level of genetic diversity among PAEs-degrading bacteria in the natural environment and their great potential to clean up phthalates-contaminated environments.

Complete degradation of di-n-octyl phthalate by biochemical cooperation between Gordonia sp. strain JDC-2 and Arthrobacter sp. strain JDC-32 isolated from activated sludge.

Two bacterial strains were isolated from activated sludge using mixtures of phthalic acid esters (PAEs) as the sole source of carbon and energy. One of the isolates was identified as Gordonia sp. strain JDC-2 and the other as Arthrobacter sp. strain JDC-32, mainly through 16S rRNA gene sequence analysis. Gordonia sp. strain JDC-2 rapidly degraded di-n-octyl phthalate (DOP) into phthalic acid (PA), which accumulated in the culture medium. Arthrobacter sp. strain JDC-32 degraded PA but not DOP. The co-culture of Gordonia sp. strain JDC-2 and Arthrobacter sp. strain JDC-32 degraded DOP completely by overcoming the degradative limitations of each species alone. The biochemical pathway of DOP degradation by Gordonia sp. strain JDC-2 was proposed based on the identified degradation intermediates. The results suggest that DOP is completely degraded by the biochemical cooperation of different microorganisms isolated from activated sludge.

[Isolation and identification of four DBP-degrading strains and molecular cloning of the degradation genes].

Four di-butyl-phthalate(DBP)-degrading bacterial strains, JDC-1, JDC-8, JDC-9 and JDC-12, were isolated from soil. The strains were gram positive. The 16S rRNA sequence analysis revealed that the four strains had similarities of 99% with Arthrobacter sp.. According to the morphologic, physiobiochemical characteristics and the analysis of their 16S rRNA, all the four strains were identified as Arthrobacter sp.. A 900 bp DNA fragment was obtained from the four strains by PCR amplified and clone. When compared with the large subunit of phthalate dioxygenase gene (phtA) of Arthrobacter keyseri, more than 96% similarities were evident in the nucleotide sequences. The optimal growth conditions and degradation rates of DBP were tested and the result indicated that the optimal growth conditions of the four bacteria strains were pH 7.0-8.5 and 30-35 degrees C. All the four bacteria strains performed efficiently for DBP degrading capabilities under optimal conditions. The most efficient strain JDC-1 degraded 500 mg/L DBP completely within 28 h whereas the least efficient strain JDC-8 degraded 500 mg/L DBP completely within 40 h. This study is helpful to the investigation of DBP-degrading mechanisms and the development of microbial resources.