Application of Co-Culture Technology to Enhance Protease Production by Two Halophilic Bacteria, Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus.

Although axenic microbial cultures form the basis of many large successful industrial biotechnologies, the production of single commercial microbial strains for use in large environmental biotechnologies such as wastewater treatment has proved less successful. This study aimed to evaluate the potential of the co-culture of two halophilic bacteria, Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus for enhanced protease activity. The co-culture was significantly more productive than monoculture (1.6-2.0 times more growth), with Marinobacter hydrocarbonoclasticus being predominant (64%). In terms of protease activity, enhanced total activity (1.8-2.4 times) was observed in the co-culture. Importantly, protease activity in the co-culture was found to remain active over a much broader range of environmental conditions (temperature 25 °C to 60 °C, pH 4-12, and 10-30% salinity, respectively). This study confirms that the co-culturing of halophilic bacteria represents an economical approach as it resulted in both increased biomass and protease production, the latter which showed activity over arange of environmental conditions.

Nanoliter scale electrochemistry of natural and engineered electroactive bacteria.

Bacterial extracellular electron transfer (EET) is envisioned for use in applied biotechnologies, necessitating electrochemical characterization of natural and engineered electroactive biofilms under conditions similar to the target application, including small-scale biosensing or biosynthesis platforms, which is often distinct from standard 100 mL-scale stirred-batch bioelectrochemical test platforms used in the laboratory. Here, we adapted an eight chamber, nanoliter volume (500 nL) electrochemical flow cell to grow biofilms of both natural (Biocathode MCL community, Marinobacter atlanticus, and Shewanella oneidensis MR1) or genetically modified (S. oneidensis ΔMtr and S. oneidensis ΔMtr + pLB2) electroactive bacteria on electrodes held at a constant potential. Maximum current density achieved by unmodified strains was similar between the nano- and milliliter-scale reactors. However, S. oneidensis biofilms engineered to activate EET upon exposure to 2,4-diacetylphloroglucinol (DAPG) produced current at wild-type levels in the stirred-batch reactor, but not in the nanoliter flow cell. We hypothesize this was due to differences in mass transport of DAPG, naturally-produced soluble redox mediators, and oxygen between the two reactor types. Results presented here demonstrate, for the first time, nanoliter scale chronoamperometry and cyclic voltammetry of a range of electroactive bacteria in a three-electrode reactor system towards development of miniaturized, and potentially high throughput, bioelectrochemical platforms.

Biochemical tests to determine the biodegradability potential of bacterial strains in PAH polluted sites.

Although the use of degrading-bacteria is one of the most efficient methods for the bioremediation of polluted sites, detection, selection and proliferation of the most efficient and competing bacteria is still a challenge. The objective of this multi-stage research was to investigate the effects of the selected bacterial strains on the degradation of anthracene, florentine, naphthalene, and oil, determined by biochemical tests. In the first stage, using the following tests: (a) biosurfactant production (emulsification, oil spreading, number of drops, drop collapse, and surface tension), (b) biofilm production, (c) activity of laccase enzyme, and (d) exopolysaccaride production, the three bacterial strains with the highest degrading potential including Bacillus pumilus, B. aerophilus, and Marinobacter hydrocarbonoclasticus were chosen. In the second stage using the following tests: (a) bacterial growth, (b) laccase enzyme activity, and (c) biosurfactant production (emulsification, oil spreading, and collapse of droplet) the degrading ability of the three selected bacterial strains plus Escherichia coli were compared. Different bacterial strains were able to degrade anthracene, florentine, naphthalene, and oil by the highest rate, three days after inoculation (DAI). However, M. hydrocarbonoclasticus showed the highest rate of florentine degradation. Although with increasing pollutant concentration the degrading potential of the bacterial strains significantly decreased, M. hydrocarbonoclasticus was determined as the most efficient bacterial strain.

Hydrophobicity-responsive engineered mesoporous silica nanoparticles: application in the delivery of essential nutrients to bacteria combating oil spills.

Facile chemical modification of mesoporous silica particles allows the production of gated reservoir systems capable of hydrophobicity-triggered release. Applied to the delivery of nutrients specifically to an oil phase, the systems developed have been shown to reliably assist the bacterial degradation of hydrocarbons. The gated system developed, made of C18 hydrocarbon chains, is demonstrated to be in a closed collapsed state in an aqueous environment, yet opens up through solvation by lipophilic alkanes and releases its content on contact with the oil phase.

A non-toxic microbial surfactant from Marinobacter hydrocarbonoclasticus SdK644 for crude oil solubilization enhancement.

This study aims to investigate the ability of a biosurfactant produced by Marinobacter hydrocarbonoclasticus strain SdK644 isolated from hydrocarbon contaminated sediment to enhance the solubilization rate of crude oil contaminated seawater. Phylogenetic analysis shows that strain SdK644 was very closely related to M. hydrocarbonoclasticus with 16S rRNA gene sequence similarity of 97.44%. Using waste frying oil as inducer carbon source, the producing biosurfactant by strain SdK644 was applied to improve crude oil solubilization in seawater. The preliminary characterization of the produced biosurfactant by FT-IR analysis indicates its possible classification in a glycolipids group. Results from crude oil solubilization assay showed that SdK644 strain biosurfactant was 2-fold greater than Tween 80 surfactant in crude oil solubilization and 12-fold higher than seawater control, as shown by GC-MS analysis of aliphatic compounds. Furthermore, this bioactive compound was shown to be nontoxic against Artemia larvae in short-term acute toxicity bioassay. Generally, the results showed the possible use of M. hydrocarbonoclasticus strain SdK644 biosurfactant in bioremediation processes of the marine environments.

Marinobacter sp. from marine sediments produce highly stable surface-active agents for combatting marine oil spills.

BACKGROUND: The application of chemical dispersants as a response to marine oil spills is raising concerns related to their potential toxicity also towards microbes involved in oil biodegradation. Hence, oil spills occurring under marine environments necessitate the application of biodispersants that are highly active, stable and effective under marine environment context. Biosurfactants from marine bacteria could be good candidates for the development of biodispersant formulations effective in marine environment. This study aimed at establishing a collection of marine bacteria able to produce surface-active compounds and evaluating the activity and stability of the produced compounds under conditions mimicking those found under marine environment context. RESULTS: A total of 43 different isolates were obtained from harbor sediments. Twenty-six of them produced mainly bioemulsifiers when glucose was used as carbon source and 16 were biosurfactant/bioemulsifiers producers after growth in the presence of soybean oil. Sequencing of 16S rRNA gene classified most isolates into the genus Marinobacter. The produced emulsions were shown to be stable up to 30 months monitoring period, in the presence of 300 g/l NaCl, at 4 °C and after high temperature treatment (120 °C for 20 min). The partially purified compounds obtained after growth on soybean oil-based media exhibited low toxicity towards V. fischeri and high capability to disperse crude oil on synthetic marine water. CONCLUSIONS: To the best of our knowledge, stability characterization of bioemulsifiers/biosurfactants from the non-pathogenic marine bacterium Marinobacter has not been previously reported. The produced compounds were shown to have potential for different applications including the environmental sector. Indeed, their high stability in the presence of high salt concentration and low temperature, conditions characterizing the marine environment, the capability to disperse crude oil and the low ecotoxicity makes them interesting for the development of biodispersants to be used in combatting marine oil spills.

The extracellular matrix of the oleolytic biofilms of Marinobacter hydrocarbonoclasticus comprises cytoplasmic proteins and T2SS effectors that promote growth on hydrocarbons and lipids.

The assimilation of the nearly water insoluble substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n-hexadecane-water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amount of proteins of unknown function that are predicted extra-cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo-proteins secreted through the type-2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n-hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates.

Marinobacter strain NNA5, a newly isolated and highly efficient aerobic denitrifier with zero N2O emission.

An efficient aerobic denitrification bacterium, strain NNA5, was isolated and identified as Marinobacter sp. NNA5. NNA5 did not perform heterotrophic nitrification. GC/IRMS analysis revealed that (15)N2 was produced from Na(15)NO2 and K(15)NO3. GC/MS and quantitative analyses showed that no N2O emission occurred when nitrite or nitrate was used as substrate. Single factor experiments indicated that optimal conditions for aerobic denitrification were: sodium succinate or sodium pyruvate as carbon source, temperature 35 °C, NaCl concentration 2-4%, C/N ratio 6-8, pH 7.5, rotation speed 150 rpm (giving dissolved oxygen concentration 6.08 mg/L), NO3(-)-N concentration ranging from 140 to 700 mg/L. NNA5 displayed highly efficient aerobic denitrifying ability, with maximal NO3(-)-N removal rate 112.8 mg/L/d. In view of its ability to perform aerobic denitrification with zero N2O emission, NNA5 has great potential for future application in aerobic denitrification processes in industrial and aquaculture wastewater treatment systems.

Chemical dispersants can suppress the activity of natural oil-degrading microorganisms.

During the Deepwater Horizon oil well blowout in the Gulf of Mexico, the application of 7 million liters of chemical dispersants aimed to stimulate microbial crude oil degradation by increasing the bioavailability of oil compounds. However, the effects of dispersants on oil biodegradation rates are debated. In laboratory experiments, we simulated environmental conditions comparable to the hydrocarbon-rich, 1,100 m deep plume that formed during the Deepwater Horizon discharge. The presence of dispersant significantly altered the microbial community composition through selection for potential dispersant-degrading Colwellia, which also bloomed in situ in Gulf deep waters during the discharge. In contrast, oil addition to deepwater samples in the absence of dispersant stimulated growth of natural hydrocarbon-degrading Marinobacter. In these deepwater microcosm experiments, dispersants did not enhance heterotrophic microbial activity or hydrocarbon oxidation rates. An experiment with surface seawater from an anthropogenically derived oil slick corroborated the deepwater microcosm results as inhibition of hydrocarbon turnover was observed in the presence of dispersants, suggesting that the microcosm findings are broadly applicable across marine habitats. Extrapolating this comprehensive dataset to real world scenarios questions whether dispersants stimulate microbial oil degradation in deep ocean waters and instead highlights that dispersants can exert a negative effect on microbial hydrocarbon degradation rates.

High sulfate reduction efficiency in a UASB using an alternative source of sulfidogenic sludge derived from hydrothermal vent sediments.

Sulfidogenesis in reactors is mostly achieved through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. In this work, an upflow anaerobic sludge blanket (UASB) reactor operated under sulfate-reducing conditions was inoculated with hydrothermal vent sediments to carry out sulfate reduction using volatile fatty acids (VFAs) as substrate and chemical oxygen demand (COD)/SO4 (-2) ratios between 0.49 and 0.64. After a short period of adaptation, a robust non-granular sludge was capable of achieving high sulfate reduction efficiencies while avoiding competence with methanogens and toxicity to the microorganisms due to high sulfide concentration. The highest sulfide concentration (2,552 mg/L) was obtained with acetate/butyrate, and sulfate reduction efficiencies were up to 98 %. A mixture of acetate/butyrate, which produced a higher yielding of HS(-), was preferred over acetate/propionate/butyrate since the consumption of COD was minimized during the process. Sludge was analyzed, and some of the microorganisms identified in the sludge belong to the genera Desulfobacterium, Marinobacter, and Clostridium. The tolerance of the sludge to sulfide may be attributed to the syntrophy among these microorganisms, some of which have been reported to tolerate high concentrations of sulfide. To the best of our knowledge, this is the first report on the analysis of the direct utilization of hydrothermal vent sediments as an alternate source of sludge for sulfate reduction under high sulfide concentrations.

Anti-biofilm performance of three natural products against initial bacterial attachment.

Marine bacteria contribute significantly towards the fouling consortium, both directly (modern foul release coatings fail to prevent "slime" attachment) and indirectly (biofilms often excrete chemical cues that attract macrofouling settlement). This study assessed the natural product anti-biofilm performance of an extract of the seaweed, Chondrus crispus, and two isolated compounds from terrestrial sources, (+)-usnic acid and juglone, against two marine biofilm forming bacteria, Cobetia marina and Marinobacter hydrocarbonoclasticus. Bioassays were developed using quantitative imaging and fluorescent labelling to test the natural products over a range of concentrations against initial bacterial attachment. All natural products affected bacterial attachment; however, juglone demonstrated the best anti-biofilm performance against both bacterial species at a concentration range between 5-20 ppm. In addition, for the first time, a dose-dependent inhibition (hormetic) response was observed for natural products against marine biofilm forming bacteria.

Marinobacter persicus sp. nov., a moderately halophilic bacterium from a saline lake in Iran.

A Gram-negative, non-endospore-forming, rod shaped, strictly aerobic, moderately halophilic bacterium, designated strain M9B(T), was isolated from the hypersaline lake Aran-Bidgol in Iran. Cells of strain M9B(T) were found to be motile and produce colonies with an orange-yellow pigment. Growth was determined to occur between 5 and 20 % (w/v) NaCl and the isolate grew optimally at 7.5-10 % (v/w) NaCl. The optimum pH and temperature for growth of the strain were determined to be pH 7.0 and 35 °C, respectively, while it was able to grow over pH and temperature ranges of 6-8 and 25-45 °C, respectively. Phylogenetic analysis based on the comparison of 16S rRNA gene sequences revealed that strain M9B(T) is a member of the genus Marinobacter. The closest relative to this strain was found to be Marinobacter hydrocarbonoclasticus MBIC 1303(T) with a similarity level of 97.7 %. DNA-DNA hybridization between the novel isolate and this phylogenetically related species was 13 ± 2 %. The major cellular fatty acids of the isolate were identified as C16:0, C19:1 ω6c, C18:1 ω9c and C16:1 ω9c. The polar lipid pattern of strain M9B(T) was determined to consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and three phospholipids. Ubiquinone 9 (Q-9) was the only lipoquinone detected. The G+C content of the genomic DNA of this strain was determined to be 58.6 mol%. Phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data suggest that this strain represents a novel species of the genus Marinobacter, for which the name Marinobacter persicus sp. nov. is proposed. The type strain of Marinobacter persicus is strain M9B(T) (=IBRC-M 10445(T) = CCM 7970(T) = CECT 7991(T) = KCTC 23561(T)).

Genome sequence of the marine bacterium Marinobacter hydrocarbonoclasticus SP17, which forms biofilms on hydrophobic organic compounds.

Marinobacter hydrocarbonoclasticus SP17 forms biofilms specifically at the interface between water and hydrophobic organic compounds (HOCs) that are used as carbon and energy sources. Biofilm formation at the HOC-water interface has been recognized as a strategy to overcome the low availability of these nearly water-insoluble substrates. Here, we present the genome sequence of SP17, which could provide further insights into the mechanisms of enhancement of HOCs assimilation through biofilm formation.

Siderophore-mediated iron uptake in two clades of Marinobacter spp. associated with phytoplankton: the role of light.

Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). We have previously shown that algal-associated heterotrophic bacteria belonging to the γ-proteobacterial Marinobacter genus release the siderophore vibrioferrin (VF). The iron-VF complex was shown to be both far more photolabile than all previously examined photolabile siderophores and to generate a photoproduct incapable of re-chelating the released iron. Thus, the photo-generated iron was shown to be highly bioavailable both to the producing bacterium and its algal partner. In exchange, we proposed that algal cells produced dissolved organic matter that helped support bacterial growth and ultimately fueled the biosynthesis of VF through a light-dependent "carbon for iron mutualism". While our knowledge of the importance of light to phototrophs is vast, there are almost no studies that examine the effects of light on microbial heterotrophs. Here, we characterize iron uptake mechanisms in "algal-associated" VF-producers. Fe uptake by a VF knock-out mutant mimics the wild-type strain and demonstrates the versatility of iron uptake mechanisms in Marinobacter VF-producers. We also show that VF-producers selectively regulate a subset of their siderophore-dependent iron uptake genes in response to light exposure. The regulation of iron uptake and transport genes by light is consistent with the light driven algal-bacterial "carbon for iron mutualism" hypothesis in the marine environment.

Anaerobic ammonium oxidation mediated by Mn-oxides: from sediment to strain level.

Nitrite and (29)N(2) productions in slurry incubations of anaerobically sediment after (15)NO(3) or (15)NH(4) labelling in the presence of Mn-oxides suggested that anaerobic Mn-oxides mediated nitrification coupled with denitrification in muddy intertidal sediments of Arcachon Bay (SW Atlantic French coast). From this sediment, bacterial strains were isolated and physiologically characterized in terms of Mn-oxides and nitrate reduction as well as potential anaerobic nitrification. One of the isolated strain, identified as Marinobacter daepoensis strain M4AY14, was a denitrifier. Nitrous oxide production by this strain was demonstrated in the absence of nitrate and with Mn-oxides and NH(4) amendment, giving indirect proof of anaerobic nitrate or nitrite production. Anaerobic Mn-oxide-mediated nitrification was confirmed by (29)N(2) production in the presence of (15)NO(3) and (14)NH(4) under denitrifying conditions. Anaerobic nitrification by M4AY14 seemed to occur only in the absence of nitrate, or at nitrate levels lower than that of Mn-oxides. Most of the other isolates were affiliated with the Shewanella genus and were able to use both nitrate and Mn-oxides as electron acceptors. When both electron acceptors were present, whatever their concentrations, nitrate and Mn-oxide reduction co-occurred. These data indicate that bacterial Mn-oxide reduction could be an important process in marine sediments with low oxygen concentrations, and demonstrate for the first time the role of bacteria in anaerobic Mn-mediated nitrification.

Biodegradation and microbial community changes upon shrimp shell wastes amended in mangrove river sediment.

Chitin, a homopolymer of N-acetyl-D-glucosamine (GlcNAc) residues linked by beta 1-4 bonds, is the most abundant renewable natural resource after cellulose. It is widely distributed in nature as the integuments of crustaceans and insects and as a component of fungi and algae. This study investigated the effects of a bifunctional chitinase/lysozyme-producing strain, Pseudomonas aeruginosa K-187, on degradation of shrimp shells and the survival conditions of bacterial strains in mangrove river sediment of Tamsui River. The structures of the whole bacterial community of the samples were measured by using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique. Results show that three bacterial strains (Acrobacter sp., Shewanella sp., and Marinobacterium sp.) which originated from the mangrove river sediment were found predominant in the 6 days-incubation sample of P. aeruginosa K-187 amended mangrove river sediment. Meanwhile, biomass, reducing sugar, and total sugar were found highest in the 6 weeks-incubation sample of shrimp shell powder and P. aeruginosa K-187-amended mangrove river sediment. According to the results, we assumed that the amendment of P. aeruginosa K-187 can enhance the biodegradation of shrimp shells in the seawater containing mangrove river sediment. We hope that these findings may provide some useful information for the reclamation of chitin-containing wastes in our environment.

Hydrostatic pressure affects membrane and storage lipid compositions of the piezotolerant hydrocarbon-degrading Marinobacter hydrocarbonoclasticus strain #5.

A new piezotolerant alkane-degrading bacterium (Marinobacter hydrocarbonoclasticus strain #5) was isolated from deep (3475 m) Mediterranean seawater and grown at atmospheric pressure (0.1 MPa) and at 35 MPa with hexadecane as sole source of carbon and energy. Modification of the hydrostatic pressure influenced neither the growth rate nor the amount of degraded hexadecane (approximately 90%) during 13 days of incubation. However, the lipid composition of the cells sharply differed under both pressure conditions. At 0.1 MPa, M. hydrocarbonoclasticus #5 biosynthesized large amounts ( approximately 62% of the total cellular lipids) of hexadecane-derived wax esters (WEs), which accumulated in the cells under the form of individual lipid bodies. Intracellular WEs were also synthesized at 35 MPa, but their proportion was half that at 0.1 MPa. This lower WE content at high pressure was balanced by an increase in the total cellular phospholipid content. The chemical composition of WEs formed under both pressure conditions also strongly differed. Saturated WEs were preferentially formed at 0.1 MPa whereas diunsaturated WEs dominated at 35 MPa. This increase of the unsaturation ratio of WEs resembled the one classically observed for bacterial membrane lipid homeostasis. Remarkably, the unsaturation ratio of membrane fatty acids of M. hydrocarbonoclasticus grown at 35 MPa was only slightly higher than at 0.1 MPa. Overall, the results suggest that intracellular WEs and phospholipids play complementary roles in the physiological adaptation of strain #5 to different hydrostatic pressures.

Proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm formation at the alkane-water interface reveals novel proteins and cellular processes involved in hexadecane assimilation.

Many hydrocarbon-degrading bacteria form biofilms at the hydrocarbon-water interface to overcome the weak accessibility of these poorly water-soluble substrates. In order to gain insight into the cellular functions involved, we undertook a proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm developing at the hexadecane-water interface. Biofilm formation on hexadecane led to a global change in cell physiology involving modulation of the expression of 576 out of 1144 detected proteins when compared with planktonic cells growing on acetate. Biofilm cells overproduced a protein encoded by MARHY0478 that contains a conserved domain belonging to the family of the outer membrane transporters of hydrophobic compounds. Homologs of MARHY0478 were exclusively found in marine bacteria degrading alkanes or possessing alkane degradation genes, and hence presumably constitute a family of alkane transporters specific to marine bacteria. Interestingly, we also found that sessile cells growing on hexadecane overexpressed type VI secretion system components. This secretion system has been identified as a key factor in virulence and in symbiotic interaction with host organisms. This observation is the first experimental evidence of the contribution of a type VI secretion system to environmental adaptation, and raises the intriguing question about the role of this secretion machine in alkane assimilation.