Oil type and temperature dependent biodegradation dynamics - Combining chemical and microbial community data through multivariate analysis.

BACKGROUND: This study investigates a comparative multivariate approach for studying the biodegradation of chemically dispersed oil. The rationale for this approach lies in the inherent complexity of the data and challenges associated with comparing multiple experiments with inconsistent sampling points, with respect to inferring correlations and visualizing multiple datasets with numerous variables. We aim to identify novel correlations among microbial community composition, the chemical change of individual petroleum hydrocarbons, oil type and temperature by creating modelled datasets from inconsistent sampling time points. Four different incubation experiments were conducted with freshly collected Norwegian seawater and either Grane and Troll oil dispersed with Corexit 9500. Incubations were conducted at two different temperatures (5 °C and 13 °C) over a period of 64 days. RESULTS: PCA analysis of modelled chemical datasets and calculated half-lives revealed differences in the biodegradation of individual hydrocarbons among temperatures and oil types. At 5 °C, most n-alkanes biodegraded faster in heavy Grane oil compared to light Troll oil. PCA analysis of modelled microbial community datasets reveal differences between temperature and oil type, especially at low temperature. For both oils, Colwelliaceae and Oceanospirillaceae were more prominent in the colder incubation (5 °C) than the warmer (13 °C). Overall, Colwelliaceae, Oceanospirillaceae, Flavobacteriaceae, Rhodobacteraceae, Alteromonadaceae and Piscirickettsiaceae consistently dominated the microbial community at both temperatures and in both oil types. Other families known to include oil-degrading bacteria were also identified, such as Alcanivoracaceae, Methylophilaceae, Sphingomonadaceae and Erythrobacteraceae, but they were all present in dispersed oil incubations at a low abundance (< 1%). CONCLUSIONS: In the current study, our goal was to introduce a comparative multivariate approach for studying the biodegradation of dispersed oil, including curve-fitted models of datasets for a greater data resolution and comparability. By applying these approaches, we have shown how different temperatures and oil types influence the biodegradation of oil in incubations with inconsistent sampling points. Clustering analysis revealed further how temperature and oil type influence single compound depletion and microbial community composition. Finally, correlation analysis of degraders community, with single compound data, revealed complexity beneath usual abundance cut-offs used for microbial community data in biodegradation studies.

Biodegradation of n-alkanes on oil-seawater interfaces at different temperatures and microbial communities associated with the degradation.

Oil biodegradation studies have mainly focused on microbial processes in dispersions, not specifically on the interfaces between the oil and the seawater in the dispersions. In this study, a hydrophobic adsorbent system, consisting of Fluortex fabrics, was used to investigate biodegradation of n-alkanes and microbial communities on oil-seawater interfaces in natural non-amended seawater. The study was performed over a temperature range from 0 to 20 °C, to determine how temperature affected biodegradation at the oil-seawater interfaces. Biodegradation of n-alkanes were influenced both by seawater temperature and chain-length. Biotransformation rates of n-alkanes decreased by reduced seawater temperature. Low rate coefficients at a seawater temperature of 0 °C were probably associated with changes in physical-chemical properties of alkanes. The primary bacterial colonization of the interfaces was predominated by the family Oceanospirillaceae at all temperatures, demonstrating the wide temperature range of these hydrocarbonoclastic bacteria. The mesophilic genus Oleibacter was predominant at the seawater temperature of 20 °C, and the psychrophilic genus Oleispira at 5 and 0 °C. Upon completion of n-alkane biotransformation, other oil-degrading and heterotrophic bacteria became abundant, including Piscirickettsiaceae (Cycloclasticus), Colwelliaceae (Colwellia), Altermonadaceae (Altermonas), and Rhodobacteraceae. This is one of a few studies that describe the biodegradation of oil, and the microbial communities associated with the degradation, directly at the oil-seawater interfaces over a large temperature interval.

Production and glucosylation of C50 and C 40 carotenoids by metabolically engineered Corynebacterium glutamicum.

The yellow-pigmented soil bacterium Corynebacterium glutamicum ATCC13032 is accumulating the cyclic C50 carotenoid decaprenoxanthin and its glucosides. Carotenoid pathway engineering was previously shown to allow for efficient lycopene production. Here, engineering of C. glutamicum for production of endogenous decaprenoxanthin as well as of the heterologous C50 carotenoids C.p.450 and sarcinaxanthin is described. Plasmid-borne overexpression of genes for lycopene cyclization and hydroxylation from C. glutamicum, Dietzia sp., and Micrococcus luteus, in a lycopene-producing platform strain constructed here, resulted in accumulation of these three C50 carotenoids to concentrations of about 3-4 mg/g CDW. Chromosomal deletion of a putative carotenoid glycosyltransferase gene cg0730/crtX in these strains entailed production of non-glucosylated derivatives of decaprenoxanthin, C.p.450, and sarcinaxanthin, respectively. Upon introduction of glucosyltransferase genes from M. luteus, C. glutamicum, and Pantoea ananatis, these hydroxylated C50 carotenoids were glucosylated. We here also demonstrate production of the C40 carotenoids ß-carotene and zeaxanthin in recombinant C. glutamicum strains and co-expression of the P. ananatis crtX gene was used to obtain glucosylated zeaxanthin. Together, our results show that C. glutamicum is a potentially valuable host for production of a wide range of glucosylated C40 and C50 carotenoids.

Metatranscriptome analysis reveals the putative venom toxin repertoire of the biofouling hydroid Ectopleura larynx.

Cnidarians thriving in biofouling communities on aquaculture net pens represent a significant health risk for farmed finfish due to their stinging cells. The toxins coming into contact with the fish, during net cleaning, can adversely affect their behavior, welfare, and survival, with a particularly serious health risk for the gills, causing direct tissue damage such as formation of thrombi and increasing risks of secondary infections. The hydroid Ectopleura larynx is one of the most common fouling organisms in Northern Europe. However, despite its significant economic, environmental, and operational impact on finfish aquaculture, biological information on this species is scarce and its venom composition has never been investigated. In this study, we generated a whole transcriptome of E. larynx, and identified its putative expressed venom toxin proteins (predicted toxin proteins, not functionally characterized) based on in silico transcriptome annotation mining and protein sequence analysis. The results uncovered a broad and diverse repertoire of putative toxin proteins for this hydroid species. Its toxic arsenal appears to include a wide and complex selection of toxin proteins, covering a large panel of potential biological functions that play important roles in envenomation. The putative toxins identified in this species, such as neurotoxins, GTPase toxins, metalloprotease toxins, ion channel impairing toxins, hemorrhagic toxins, serine protease toxins, phospholipase toxins, pore-forming toxins, and multifunction toxins may cause various major deleterious effects in prey, predators, and competitors. These results provide valuable new insights into the venom composition of cnidarians, and venomous marine organisms in general, and offer new opportunities for further research into novel and valuable bioactive molecules for medicine, agronomics and biotechnology.

A Novel Biosurfactant-Based Oil Spill Response Dispersant for Efficient Application under Temperate and Arctic Conditions.

Synthetic oil spill dispersants have become essential in offshore oil spill response strategies. However, their use raises significant concerns regarding toxicity to phyto- and zooplankton and other marine organisms, especially in isolated and vulnerable areas such as the Arctic and shorelines. Sustainable alternatives may be developed by replacing the major active components of commercial dispersants with their natural counterparts. During this study, interfacial properties of different types of glycolipid-based biosurfactants (rhamnolipids, mannosylerythritol lipids, and trehalose lipids) were explored in a crude oil-seawater system. The best-performing biosurfactant was further mixed with different nontoxic components of Corexit 9500A, and the interfacial properties of the most promising dispersant blend were further explored with various types of crude oils, weathered oil, bunker, and diesel fuel in natural seawater. Our findings indicate that the most efficient dispersant formulation was achieved when mannosylerythritol lipids (MELs) were mixed with Tween 80 (T). The MELs-T dispersant blend significantly reduced the interfacial tension (IFT) of various crude oils in seawater with results comparable to those obtained with Corexit 9500A. Importantly, no leaching or desorption of MELs-T components from the crude oil-water interface was observed. Furthermore, for weathered and more viscous asphaltenic bunker fuel oil, IFT results with the MELs-T dispersant blend surpassed those obtained with Corexit 9500A. This dispersant blend also demonstrated effectiveness at different dosages (dispersant-to-oil ratio (DOR)) and under various temperature conditions. The efficacy of the MELs-T dispersant was further confirmed by standard baffled flask tests (BFTs) and Mackay-Nadeau-Steelman (MNS) tests. Overall, our study provides promising data for the development of effective biobased dispersants, particularly in the context of petroleum exploitation in subsea resources and transportation in the Arctic.

Analysis and manipulation of aspartate pathway genes for L-lysine overproduction from methanol by Bacillus methanolicus.

We investigated the regulation and roles of six aspartate pathway genes in L-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by L-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the L-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has-in addition to a hom-1 mutation-chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for L-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased L-lysine production levels.

Continuous control of the flow in biochemical pathways through 5' untranslated region sequence modifications in mRNA expressed from the broad-host-range promoter Pm.

The inducible Pm promoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in the Pm DNA region corresponding to the 5' untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding ß-lactamase, luciferase, and phosphoglucomutase) in Escherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter, P1 (P(antitet)), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C(50) carotenoid sarcinaxanthin in an engineered strain of E. coli that produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing three Micrococcus luteus derived genes from the promoter Pm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.

Absolute quantification of priority bacteria in aquaculture using digital PCR.

Modern aquaculture systems are designed for intensive rearing of fish or other species. Both land-based and offshore systems typically contain high loads of biomass and the water quality in these systems is of paramount importance for fish health and production. Microorganisms play a crucial role in removal of organic matter and nitrogen-recycling, production of toxic hydrogen sulfide (H2S), and can affect fish health directly if pathogenic for fish or exerting probiotic properties. Methods currently used in aquaculture for monitoring certain bacteria species numbers still have typically low precision, specificity, sensitivity and are time-consuming. Here, we demonstrate the use of Digital PCR as a powerful tool for absolute quantification of sulfate-reducing bacteria (SRB) and major pathogens in salmon aquaculture, Moritella viscosa, Yersinia ruckeri and Flavobacterium psychrophilum. In addition, an assay for quantification of Listeria monocytogenes, which is a human pathogen bacterium and relevant target associated with salmonid cultivation in recirculating systems and salmon processing, has been assessed. Sudden mass mortality incidents caused by H2S produced by SRB have become of major concern in closed aquaculture systems. An ultra-sensitive assay for quantification of SRB has been established using Desulfovibrio desulfuricans as reference strain. The use of TaqMan® probe technology allowed for the development of multi-plex assays capable of simultaneous quantification of these aquaculture priority bacteria. In single-plex assays, limit of detection was found to be at around 20 fg DNA for M. viscosa, Y. ruckeri and F. psychrophilum, and as low as 2 fg DNA for L. monocytogenes and D. desulfuricans.

The influences of phytoplankton species, mineral particles and concentrations of dispersed oil on the formation and fate of marine oil-related aggregates.

The formation and fallout of oil-related marine snow have been associated with interactions between dispersed oil and small marine particles, like phytoplankton and mineral particles. In these studies, the influences of phytoplankton species, mineral particle concentration, and oil concentration on the aggregation of oil in seawater (SW) were investigated. The experiments were performed in a low-turbidity carousel incubation system, using natural SW at 13 °C. Aggregation was measured by silhouette camera analyses, and oil compound group distribution and depletion by gas chromatography (GC-FID or GC-MS). Aggregates with median sizes larger than 500 µm in diameter were measured in the presence of dispersed oil and the phytoplankton species Thalassiosira rotula, Phaeocystis globosa, Skeletonema pseudocostatum, but not with the microalgae Micromonas pusilla. When mineral particles (diatomaceous earth) were incubated at different concentrations (5-30 mg/L) with dispersed oil and S. pseudocostatum, the largest aggregates were measured at the lower mineral particle concentration (5 mg/L). Since dispersed oil rapidly dilutes in the marine water column, experiments were performed with oil concentrations of from 10 mg/L to 0.01 mg/L in the presence of S. pseudocostatum and diatomaceous earth. Aggregates larger than 500 µm was measured only at the highest oil concentrations (10 mg/L). However, oil attachment to the marine particles were also measured at low oil concentrations (≤1 mg/L). Depletion of oil compound groups (n-alkanes, naphthalenes, PAHs, decalins) were measured at all oil concentrations, both in aggregate and water phases, with biodegradation as the expected main depletion process. These results showed that oil concentration may be important for oil-related marine snow formation, but that even oil droplets at low concentrations may attach to the particles and be transported by prevailing currents.

Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: heterologous expression and evidence for diverse and multiple catalytic functions of C(50) carotenoid cyclases.

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for L-lysine production from methanol at 50 degrees C.

We here present the pyc gene encoding pyruvate carboxylase (PC), and the hom-1 and hom-2 genes encoding two active homoserine dehydrogenase (HD) proteins, in methylotrophic Bacillus methanolicus MGA3. In general, both PC and HD are regarded as key targets for improving bacterial L-lysine production; PC plays a role in precursor oxaloacetate (OAA) supply while HD controls an important branch point in the L-lysine biosynthetic pathway. The hom-1 and hom-2 genes were strongly repressed by L-threonine and L-methionine, respectively. Wild-type MGA3 cells secreted 0.4 g/l L-lysine and 59 g/l L-glutamate under optimised fed batch methanol fermentation. The hom-1 mutant M168-20 constructed herein secreted 11 g/l L-lysine and 69 g/l of L-glutamate, while a sixfold higher L-lysine overproduction (65 g/l) of the previously constructed classical B. methanolicus mutant NOA2#13A52-8A66 was accompanied with reduced L-glutamate production (28 g/l) and threefold elevated pyc transcription level. Overproduction of PC and its mutant enzyme P455S in M168-20 had no positive effect on the volumetric L-lysine yield and the L-lysine yield on methanol, and caused significantly reduced volumetric L-glutamate yield and L: -glutamate yield on methanol. Our results demonstrated that hom-1 represents one key target for achieving L-lysine overproduction, PC activity plays an important role in controlling L-glutamate production from methanol, and that OAA precursor supply is not a major bottleneck for L-lysine overproduction by B. methanolicus.

Multiplex droplet digital PCR assay for detection of Flavobacterium psychrophilum and Yersinia ruckeri in Norwegian aquaculture.

We report the development of ddPCR assays for single and simultaneous detection of the bacterial pathogens Flavobacterium psychrophilum and Yersinia ruckeri in water from land-based recirculation aquaculture systems (RAS), producing Atlantic salmon (Salmo salar) smolt. The method was tested and verified for use in water analyses from RAS production sites, and proved to be specific and with sensitivity 0.0011 ng DNA for F. psychrophilum and 1.24 ng for Y. ruckeri. These bacteria are important fish pathogens that have caused reoccurring salmonid infection disease in RAS. Monitoring pathogen levels in water samples could be a useful alternative surveillance strategy to evaluate operational risk assessment connected to stress factors. Water quality is essential for fish health and growth in RAS production in general, and high or increasing levels of these pathogens in the RAS water may generate an early indication of unfavourable conditions in the RAS environment, and give directions to operational actions. This approach may reduce fish mortality, reduce production loss, and offer more effective and targeted preventive measures within RAS production.

Formation and fate of oil-related aggregates (ORAs) in seawater at different temperatures.

In this study, the formation and fate of oil-related aggregates (ORAs) from chemically dispersed oil in seawater (SW) were investigated at different temperatures (5 °C, 13 °C, 20 °C). Experiments in natural SW alone, and in SW amended with typical marine snow constituents (phytoplankton and mineral particles), showed that the presence of algae stimulated the formation of large ORAs, while high SW temperature resulted in faster aggregate formation. The ORAs formed at 5 °C and 13 °C required mineral particles for sinking, while the aggregates also sank in the absence of mineral particles at 20°. Early in the experimental periods, oil compound accumulation in ORAs was faster than biodegradation, particularly in aggregates with algae, followed by rapid biodegradation. High abundances of bacteria associated with hydrocarbon biodegradation were determined in the ORAs, together with algae-associated bacteria, while clustering analyses showed separation between bacterial communities in experiments with oil alone and oil with algae/mineral particles.

Response of bacterial communities in Barents Sea sediments in case of a potential CO2 leakage from carbon reservoirs.

Carbon capture and storage sites in Barents Sea shelf are currently in progress as part of climate change mitigation activities. However environmental impacts of a possible CO2 seepage on bacterial community are lacking knowledge. This work addressed potential consequences on bacterial communities from Snøvit region in Barents Sea sediments. Long-term experiment (92 days) was carried out mimicking realistic conditions of pressure (∼30 bars) using the unique hyperbaric chamber (Karl Erik TiTank). The experiment was divided in three stages: i) 21 days of no CO2, ii) 50 days of simulation of carbon dioxide leakage (depletion of pH to 7.0) and iii) 14 days emulating a leakage cessation. Results suggested that bacterial communities can adapt to a CO2 leakage in short term. However, bacteria showed negative effects in terms of activity, community structure, and number of cells after long term CO2 exposure. After CO2 leakage cessation, bacterial communities did not show a significant recovery. These findings highlighted that, even though marine bacteria showed adaptation to the new conditions (acidified environment), in case of a small but continuous CO2 leakage marine bacteria might not be recovered upon pre-exposure status.

Dispersibility and biotransformation of oils with different properties in seawater.

Dispersants are used to remove oils slicks from sea surfaces and to generate small oil-droplet dispersions, which may result in enhanced biodegradation of the oil. In this study, dispersibility and biodegradation of chemically dispersed oils with different physical-chemical properties (paraffinic, naphthenic and asphaltenic oils) were compared in natural temperate SW at 13 °C. All selected oils were chemically dispersible when well-known commercial dispersants were used. However, interfacial tension (IFT) studies of the dispersed oils showed different IFT properties of the oils at 13 °C, and also different leaching of the dispersants from oil droplet surfaces. Biodegradation studies of the chemically dispersed oils were performed in a carousel system, with initial median droplet sizes <30 µm and oil concentrations of 2.5-2.8 mg/L. During biodegradation, oil droplet concentrations were rapidly reduced, in association with the emergence of macroscopic 'flocs'. Biotransformation results showed that half-lives of semivolatile total extractable organic carbon (TEOC), single target 2- to 4-ring PAH, and 22 oil compound groups used as input data in the oil spill contingency model OSCAR, did not differ significantly between the oils (P > 0.05), while n-alkanes half-lives differed significantly (P < 0.05). Biotransformation was associated with rapid microbial growth in all oil dispersions, in association with n-alkane and PAH biotransformation. These results have implications for the predictions of biodegradation of oil slicks treated with dispersants in temperate SW.

Biodegradation of dispersed oil in seawater is not inhibited by a commercial oil spill dispersant.

Chemical dispersants are well-established as oil spill response tools. Several studies have emphasized their positive effects on oil biodegradation, but recent studies have claimed that dispersants may actually inhibit the oil biodegradation process. In this study, biodegradation of oil dispersions in natural seawater at low temperature (5°C) was compared, using oil without dispersant, and oil premixed with different concentrations of Slickgone NS, a widely used oil spill dispersant in Europe. Saturates (nC10-nC36 alkanes), naphthalenes and 2- to 5-ring polycyclic aromatic hydrocarbons (PAH) were biotransformed at comparable rates in all dispersions, both with and without dispersant. Microbial communities differed primarily between samples with or without oil, and they were not significantly affected by increasing dispersant concentrations. Our data therefore showed that a common oil spill dispersant did not inhibit biodegradation of oil at dispersant concentrations relevant for response operations.

Microbial communities in seawater from an Arctic and a temperate Norwegian fjord and their potentials for biodegradation of chemically dispersed oil at low seawater temperatures.

Biodegradation of chemically dispersed oil at low temperature (0-2 °C) was compared in natural seawater from Arctic (Svalbard) and a temperate (Norway) fjords. The oil was premixed with a dispersant (Corexit 9500) and small-droplet oil dispersions prepared. Faster biotransformation of n-alkanes in the Arctic than in the temperate seawater were associated with the initially higher abundance of the alkane-degrading genus Oleispira in the Arctic than the temperate seawater. Comparable transformation of aromatic hydrocarbons was further associated with the late emergences Cycloclasticus in both seawater sources. The results showed that chemically dispersed oil may be rapidly biodegraded by microbial communities in Arctic seawater. Compared to oil biodegradation studies at higher seawater temperatures, longer lag-periods were experienced here, and may be attributed to both microbial and oil properties at these low seawater temperatures.

Petroleum hydrocarbon and microbial community structure successions in marine oil-related aggregates associated with diatoms relevant for Arctic conditions.

Oil-related aggregates (ORAs) may contribute to the fate of oil spilled offshore. However, our understanding about the impact of diatoms and associated bacteria involved in the formation of ORAs and the fate of oil compounds in these aggregates is still limited. We investigated these processes in microcosm experiments with defined oil dispersions in seawater at 5 °C, employing the Arctic diatom Fragilariopsis cylindrus and its associated bacterial assemblage to promote ORA formation. Accumulation of oil compounds, as well as biodegradation of naphthalenes in ORAs and corresponding water phases, was enhanced in the presence of diatoms. Interestingly, the genus Nonlabens was predominating the bacterial communities in diatom-supplemented microcosms, while this genus was not abundant in other samples. This work elucidates the relevance of diatom biomass for the formation of ORAs, microbial community structures and biodegradation processes in chemically dispersed oil at low temperatures relevant for Arctic conditions.

Microbial community and metagenome dynamics during biodegradation of dispersed oil reveals potential key-players in cold Norwegian seawater.

Oil biodegradation as a weathering process has been extensively investigated over the years, especially after the Deepwater Horizon blowout. In this study, we performed microcosm experiments at 5 °C with chemically dispersed oil in non-amended seawater. We link biodegradation processes with microbial community and metagenome dynamics and explain the succession based on substrate specialization. Reconstructed genomes and 16S rRNA gene analysis revealed that Bermanella and Zhongshania were the main contributors to initial n-alkane breakdown, while subsequent abundances of Colwellia and microorganisms closely related to Porticoccaceae were involved in secondary n­alkane breakdown and beta­oxidation. Cycloclasticus, Porticoccaceae and Spongiiabcteraceae were associated with degradation of mono- and poly-cyclic aromatics. Successional pattern of genes coding for hydrocarbon degrading enzymes at metagenome level, and reconstructed genomic content, revealed a high differentiation of bacteria involved in hydrocarbon biodegradation. A cooperation among oil degrading microorganisms is thus needed for the complete substrate transformation.

l-lysine production by Bacillus methanolicus: Genome-based mutational analysis and l-lysine secretion engineering.

Bacillus methanolicus is a methylotrophic bacterium with an increasing interest in academic research and for biotechnological applications. This bacterium was previously applied for methanol-based production of l-glutamate, l-lysine and the five-carbon diamine cadaverine by wild type, classical mutant and recombinant strains. The genomes of two different l-lysine secreting B. methanolicus classical mutant strains, NOA2#13A52-8A66 and M168-20, were sequenced. We focused on mutational mapping in genes present in l-lysine and other relevant amino acid biosynthetic pathways, as well as in the primary cell metabolism important for precursor supply. In addition to mutations in the aspartate pathway genes dapG, lysA and hom-1, new mutational target genes like alr, proA, proB1, leuC, odhA and pdhD were identified. Surprisingly, no mutations were found in the putative l-lysine transporter gene lysEMGA3. Inspection of the wild type B. methanolicus strain PB1 genome sequence identified two homologous putative l-lysine transporter genes, lysEPB1 and lysE2PB1. The biological role of these putative l-lysine transporter genes, together with the heterologous l-lysine exporter gene lysECg from Corynebacterium glutamicum, were therefore investigated. Our results demonstrated that the titer of secreted l-lysine in B. methanolicus was significantly increased by overexpression of lysECg while overexpression of lysEMGA3, lysEPB1 and lysE2PB1 had no measurable effect.