Marinobacter Salsuginis Research Articles

Formulation of Bio-Based Washing Agent and Its Application for Removal of Petroleum Hydrocarbons From Drill Cuttings Before Bioremediation.

Drill cuttings from petroleum exploration and production sites can cause diverse environmental problems. Total petroleum hydrocarbons (TPHs) are a major pollutant from the use of polyolefin-based mud. As an alternative to incineration, this study investigated the application of surfactant-enhanced washing technology prior to bioremediation. The washing step was necessary because the initial TPH concentrations were quite high at approximately 15% (w/w). Washing agents were formulated by varying the concentration of lipopeptide biosurfactant (in foamate or cell-free broth), Dehydol LS7TH (fatty alcohol ethoxylate 7EO, oleochemical surfactant) and butanol (as a lipophilic linker) at different salinities. The most efficient formula produced a Winsor Type I microemulsion (oil-in-water microemulsion) with polyolefin and contained only 20% (v/v) foamate and 2% (v/v) Dehydol LS7TH in water. Due to the synergistic behavior between the anionic lipopeptides and non-ionic Dehydol LS7TH, the formula efficiently removed 92% of the TPHs from the drill cuttings when applied in a jar test. To reduce the cost, the concentrations of each surfactant should be reduced; thus, the formula was optimized by the simplex lattice mixture design. In addition, cell-free broth, at a pH of 10, containing 3.0 g/L lipopeptides was applied instead of foamate because it was easy to prepare. The optimized formula removed 81.2% of the TPHs and contained 72.0% cell-free broth and 1.4% Dehydol LS7TH in water. A 20-kg soil washing system was later tested where the petroleum removal efficiency decreased to 70.7% due to polyolefin redeposition during separation of the washing solution. The remaining TPHs (4.5%) in the washed drilled cuttings were further degraded by a mixture of Marinobacter salsuginis RK5, Microbacterium saccharophilum RK15 and Gordonia amicalis JC11. To promote TPH biodegradation, biochar and fertilizer were applied along with bacterial consortia in a microcosm experiment. After 49-day incubation, the TPHs were reduced to 0.9% by both physical and biological mechanisms, while the TPHs in the unamended samples remained unaffected. With the use of the formulated bio-based washing agent and bioremediation approach, the on-site treatment of drill cuttings could be conducted with an acceptable cost and low environmental impacts.

Isolation and characterization of a moderately halophilic Marinobacter phage-host system from the Arabian Sea.

Marinobacter is an ecologically important genus of Gammaproteobacteria found in diverse marine habitats, many species of which are capable of degrading hydrocarbons. In this study, we isolated a Marinobacter phage-host system from the surface waters of the Arabian Sea using enrichment culture methods, studied their growth characteristics and investigated the effect of salinity and nitrate concentrations on phage-host interactions. The bacterial isolate had maximum identity to Marinobacter salsuginis based on 16S rRNA similarities and was termed as Marinobacter sp., strain D1S9. It could tolerate up to 14% of NaCl with maximum growth at 11% NaCl. The host grew optimally between 35 and 40°C and at pH8. It had a generation time of 3.7h with a mean growth rate of 0.27h-1. The phage infected the host forming clear, round plaques of 1-2mm diameter. It had a narrow host range restricted to the strain Marinobacter D1S9. The latent period and burst size of the phage were estimated to be 30min and 106 phages per infected cell, respectively. The phage had an adsorption rate of 3.4 × 10-8mlmin-1 and retained 40.4% of its adsorption efficiency at 16% NaCl with a maximum at 4% NaCl (76.1%). Inorganic nitrate was found to have a direct role in controlling host growth and phage burst size.

An induced corrosion inhibition of X80 steel by using marine bacterium Marinobacter salsuginis

The corrosion behaviour of X80 pipeline steel was studied in a simulated marine environment inoculated with marine bacterium Marinobacter salsuginis. The electrochemical results showed that the increase in linear polarization resistance, charge transfer resistance, and the decrease in corrosion current density of the X80 pipeline steel immersed in the biotic medium indicated its high corrosion resistance compared to those in the abiotic medium. Surface morphological techniques including scanning electron microscopy, confocal laser scanning microscopy and live/dead cells staining were employed to observe the biofilm morphology and bacterial viability after different immersion times. X-ray photoelectron spectroscopy was used to analyse the oxides film formed on the steel surface. The obtained results indicated that the corrosion inhibition efficiency was obviously higher in the biotic medium compared to that in the abiotic medium. The high corrosion resistance of X80 steel in biotic medium was attributed to the formation of biofilm and the development of extracellular polymeric substances (EPS) layer on its surface.

Aerobic degradation of cis-dichloroethene by the marine bacterium Marinobacter salsuginis strain 5N-3.

An aerobic bacterium, designated strain 5N-3 (NBRC 113055), that degrades cis-dichloroethene (cDCE) was isolated from a sea sediment in Japan. Strain 5N-3 was able to degrade a certain amount of cDCE in the presence of pyruvate without the action of inducers. In the presence of inducers, such as phenol and benzene, the strain completely removed cDCE. By the application of 16S ribosomal RNA (16S rRNA) gene sequencing and average nucleotide identity analyses, the strain 5N-3 was identified as Marinobacter salsuginis. On the other hand, identified species of Marinobacter are not known to degrade cDCE at all. A draft genome sequence analysis of the strain 5N-3 suggested that the dmp-homologous operon (operon for phenol degradation) may be contributing to the aerobic degradation of cDCE. This is the first report on an aerobic marine bacterium that has been found to degrade cDCE.

Marinobacter maroccanus sp. nov., a moderately halophilic bacterium isolated from a saline soil.

During the taxonomic investigation of exopolymer-producing halophilic bacteria, a rod-shaped, motile, Gram-stain-negative, halophilic bacterium, designated strain N4T, was isolated from a saline soil located in northern Morocco. Optimal growth of the isolate was at 30-37 ºC and at pH 7.0-8.0, in the presence of 5-7 % (w/v) NaCl. Useful characteristics for the phenotypic differentiation of strain N4T from other Marinobacter species included α-chymotrypsin and α-glucosidase activities and the carbohydrate assimilation profile. The major fatty acids detected in strain N4T were C16:0 and C18:1ω9c and the predominant respiratory quinone was ubiquinone-9. Sequence analysis of the 16S rRNA gene indicated that strain N4T belonged to the genus Marinobacter and was closely related to the type strains of Marinobacter adhaerens (99.04 % similarity), Marinobacter salsuginis (98.97 %) and Marinobacter flavimaris (98.36 %). Phylogenetic analysis of the rpoD gene sequence also showed that the nearest neighbours of strain N4T were M. salsuginis (91.49 % similarity), M. adhaerens and M. flavimaris (90.63 %). Strain N4T showed 87.98 % average nucleotide identity with M. flavimaris and M. salsuginis, and 87.47 % with M. adhaerens. Regarding in-silico genome-to-genome distance, strain N4T showed DNA-DNA hybridization values of 33.30 % with M. adhaerens, 34.60 % with M. flavimaris and 34.70 % with M. salsuginis. The DNA G+C content of strain N4T was 57.3 mol%. Based on the results of phenotypic characterization, phylogenetic analysis and genome comparison, strain N4T represents a novel species of the genus Marinobacter, for which the name Marinobacter maroccanus sp. nov. is proposed. The type strain is N4T (=CECT 9525T=LMG 30466T).

ISOLATION AND SCREENING OF BIOSURFACTANT-PRODUCING MARINE BACTERIA FROM KUANTAN PORT, PAHANG, MALAYSIA

Biosurfactants play an important role in bioremediation of organic pollutants such as petroleum hydrocarbon. The unique properties of biosurfactants make them possible to be used in the remediation of hydrocarbon contaminated sites. Therefore, the existence of indigenous microorganisms that have the ability to consume petroleum hydrocarbon as carbon source and simultaneously produce biosurfactants in order to facilitate the hydrocarbon metabolism can be manipulated for bioremediation purposes. In this study, isolation and screening of potential biosurfactant-producing bacteria from two sampling points in Kuantan Port seawater were successfully done. Amongst the isolates, 4 out of 7 isolates from Point A were Gram negative bacteria and 2 out 5 isolates from Point B were Gram negative bacteria. The positive oxidase test resulted for all isolates from Point A and only B5 from Point B produced negative result. Catalase test conducted produced positive results on isolates from Point A (A3, A5, A6& A7) and Point B (B1, B2, B4 & B5).The highest percentage emulsification index measured belonged to isolate B4 and B5 which are 67%, thus make these isolates to be the most promising biosurfactant producers. Further identification by 16S rRNA gene found that isolates were closely related to Rhodococcus erythropolis (A1), Psedomonas stutzeri (A2), Pseudoalteromonas lipolytica (A3, A6 and B4), Vibrio brasiliensis (A4 and B2), Vibrio tubiashii (B1), Marinobacter salsuginis (A5), Labrenzia aggregate (A7), Marinococcus halophilus (B3) and Thalassospira xianmenensis(B5). Hence, through biosurfactant activities exhibited by isolates, B4 and B5 were the most potential isolates to produce biosurfactant. Therefore, these isolates can potentially be exploited to aid in bioremediation of petroleum hydrocarbon contaminated sites and would also be useful to enhance oil recovery in petroleum industry.

Marinobacter salinus sp. nov., a moderately halophilic bacterium isolated from a tidal flat environment.

Two Gram-stain-negative, aerobic, motile, halophilic, rod-shaped bacteria, designated Hb8T and Hb20, were isolated from a tidal flat environment located on the South-West Korean peninsula. The isolates grew at 10-37 °C, at pH 5.0-9.0 and in NaCl concentrations of 0.5-15 % (w/v; optimum, 3.0-6.0 %). Sequence analysis of the 16S rRNA indicated that the isolates belong to the genus Marinobacter and are most closely related to Marinobacter sediminumR65T (98.3 %), followed by Marinobacter lipolyticus SM19T, Marinobacter salsuginis SD-14BT and Marinobacter similis A3d10T. The overall 16S rRNA gene sequence similarity with these species was 97.9 %, but Hb8T and Hb20 showed 100 % sequence similarity with each other. DNA-DNA relatedness values of H8T and Hb20 suggested that these isolates represent a single species, while DNA-DNA relatedness values of the two novel isolates with M. sediminum DSM 27079T and M. similis DSM 15400T were only 21.3 and 22.9 %, respectively. The major fatty acids present in strain Hb8T were identified as C16 : 0, C16 : 1ω9c, C18 : 1ω9c, C18 : 0 3-OH and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). Ubiquinone-9 was the main respiratory quinone in both the novel strains. The polar lipids found to be present included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified phospholipids and five unidentified lipids. The genomic DNA G+C content of Hb8T and Hb20 was 54.5 mol%. Polyphasic analysis indicated that the two isolates are representatives of a novel species of the genus Marinobacte, for which the name Marinobacter salinus sp. nov. is proposed. The type strain is Hb8T (=KCTC 52255T=JCM 31416T).

Marinobacter halotolerans sp. nov., a halophilic bacterium isolated from a saltern crystallizing pond.

A Gram-stain-negative, moderately halophilic, motile bacterium, designated strain CP12T, was isolated from a crystallizing pond of a saltern of the Yellow Sea in Korea. Cells of strain CP12T were non-spore-forming rods and produced whitish-yellow colonies. Growth was observed at 10-37 °C (optimum 37 °C), at pH 6.0-9.0 (optimum pH 7.0), and in the presence of 0.5-20 % (w/v) NaCl (optimum 3 %). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CP12T was closely related to Marinobacter flavimaris SW-145T (98.4 % 16S rRNA gene sequence similarity), Marinobacter algicola DG893T (98.2 %), Marinobacter adhaerens HP15T (98.2 %), Marinobacter salsuginis SD-14BT (97.9 %), Marinobacter salarius R9SW1T (97.6 %) and Marinobacter lipolyticus SM19T (97.1 %). DNA-DNA hybridization studies showed values lower than 18.6 % between strain CP12T and any of these species. The predominant respiratory isoprenoid quinone was ubiquinone-9 and the major cellular fatty acids of strain CP12T were C16 : 0, C12 : 0 3-OH, C12 : 0, Summed feature 3, C16 : 0 10-methyl and C18 : 1ω9c. On the basis of phenotypic properties, and phylogenetic and chemotaxonomic data, it is evident that strain CP12T represents a novel species of the genus Marinobacter, for which the name Marinobacter halotolerans sp. nov. is proposed. The type strain is CP12T (=KACC 18381T=NBRC 110910T).

Marinobacter confluentis sp. nov., a lipolytic bacterium isolated from a junction between the ocean and a freshwater lake.

A Gram-stain-negative, motile, aerobic and rod-shaped bacterium, designated HJM-18T, was isolated from the place where the ocean and a freshwater lake meet at Hwajinpo, South Korea, and subjected to a taxonomic study using a polyphasic approach. Strain HJM-18T grew optimally at 30 °C, at pH 7.0-8.0 and in the presence of 1.0-3.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences showed that strain HJM-18T belonged to the genus Marinobacter. Strain HJM-18T exhibited 16S rRNA gene sequence similarity values of 97.05-98.22 % to the type strains of Marinobacter algicola, Marinobacter flavimaris, Marinobacter adhaerens, Marinobacter salarius, Marinobacter salsuginis, Marinobacter guineae and Marinobacter gudaonensis and of 93.21-96.98 % to the type strains of the other species of the genus Marinobacter. Strain HJM-18T contained Q-9 as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C18 : 1ω9c as the major fatty acids. The major polar lipids detected in strain HJM-18T were phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminophospholipid. The DNA G+C content was 58 mol% and the mean DNA-DNA relatedness values with the type strains of the seven phylogenetically related species of the genus Marinobacter were 10-27 %. Differential phenotypic properties, together with phylogenetic and genetic distinctiveness, revealed that strain HJM-18T is separated from recognized species of the genus Marinobacter. On the basis of the data presented, strain HJM-18T represents a novel species of the genus Marinobacter, for which the name Marinobacter confluentis sp. nov. is proposed. The type strain is HJM-18T ( = KCTC 42705T = NBRC 111223T).

Use of a Noctiluca-killing bacterium Marinobacter salsuginis strain BS2 to reduce shrimp mortality caused by Noctiluca scintillans

The ability of an algicidal bacterium Marinobacter salsuginis strain BS2, isolated from shrimp pond water, to reduce shrimp mortality was investigated under laboratory conditions. When two species of shrimp (Penaeus monodon and Litopenaeus vannamei) (body length 1.5–1.8 cm) were cultured together with the dinoflagellate Noctiluca scintillans, nearly 80 % of the shrimps died within 7 days. However, when bacterial strain BS2 was also added to the culture, N. scintillans was killed within 48 h, and shrimp survival rates on the 7th day improved from 23 to 87 % for both P. monodon and L. vannamei. The bacterium BS2 alone had no effect on shrimp condition. Under conditions of increased dissolved oxygen, the effect of using BS2 was greater, and shrimp survival rates improved even more dramatically, from 26 to 98 %. These studies provide the first evidence that the use of killing bacteria, isolated from shrimp culture water, can suppress harmful algal blooms (HABs) and thus restore the efficiency of shrimp production. The control of HABs in this way in shrimp culture farms would be a major benefit for shrimp production.

Marinobacter adhaerens sp. nov., isolated from marine aggregates formed with the diatom Thalassiosira weissflogii

A Gram-negative, motile, rod-shaped bacterial strain, HP15(T), was isolated from aggregates taken from surface waters of the German Wadden Sea (German Bight). Of 82 marine isolates, HP15(T) was chosen for further study because of its high potential to induce production of transparent exopolymeric particles and aggregate formation while interacting with the diatom Thalassiosira weissflogii. HP15(T) grew optimally at 34-38 °C and pH 7.0-8.5, and was able to tolerate salt concentrations of 0.5-20% (w/v) NaCl. HP15(T) was characterized chemotaxonomically by possessing ubiquinone-9 as the major respiratory lipoquinone, as well as C(16:0), C(18:1)ω9c and C(16:1)ω7c/iso-C(15:0) 2-OH as the predominant fatty acids. The DNA G+C content of strain HP15(T) was 56.9 mol%. The closest relative based on 16S rRNA gene sequence analysis was the type strain of Marinobacter flavimaris, with 99% similarity. Whole-genome relatedness values of HP15(T) to the type strains of M. flavimaris, Marinobacter salsuginis, Marinobacter lipolyticus and Marinobacter algicola were less than 70%, as determined by DNA-DNA hybridization. On the basis of phenotypic and chemotaxonomic properties as well as phylogenetic analyses, the isolate represents a novel species, Marinobacter adhaerens sp. nov.; the type strain is HP15(T) (=DSM 23420(T)=CIP 110141(T)).

Marinobacter zhanjiangensis sp. nov., a marine bacterium isolated from sea water of a tidal flat of the South China Sea

A novel Gram-negative, catalase- and oxidase-positive, non-sporulating, rod-shaped, aerobic bacterium, designated strain JSM 078120(T), was isolated from sea water collected from a tidal flat of Naozhou Island, South China Sea. Growth occurred with 1-15% (w/v) total salts (optimum, 2-4%), at pH 6.0-10.0 (optimum, pH 7.5) and at 4-35 degrees C (optimum, 25-30 degrees C). The major cellular fatty acids were C(18:1) omega9c, C(16:0), C(12:0) 3-OH and C(16:1) omega7c. The predominant respiratory quinone was ubiquinone Q-9, and the genomic DNA G + C content was 60.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 078120(T) should be assigned to the genus Marinobacter, being related most closely to the type strains of Marinobacter segnicrescens (sequence similarity 98.2%), Marinobacter bryozoorum (97.9%) and Marinobacter gudaonensis (97.6%). The sequence similarities between the novel isolate and the type strains of other recognized Marinobacter species ranged from 96.7 (with Marinobacter salsuginis) to 93.3% (with Marinobacter litoralis). The levels of DNA-DNA relatedness between strain JSM 078120(T) and the type strains of M. segnicrescens, M. bryozoorum and M. gudaonensis were 25.3, 20.6 and 18.8%, respectively. The combination of phylogenetic analysis, DNA-DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 078120(T) represents a novel species of the genus Marinobacter, for which the name Marinobacter zhanjiangensis sp. nov. is proposed. The type strain is JSM 078120(T) (= CCTCC AB 208029(T) = DSM 21077(T) = KCTC 22280(T)).

Marinobacter salsuginis sp. nov., isolated from the brine–seawater interface of the Shaban Deep, Red Sea

Two moderately halophilic Gram-negative bacteria were isolated from a sample taken from the brine-seawater interface of the Shaban Deep in the Red Sea. Phylogenetic analysis of the 16S rRNA gene sequence showed that these organisms represent a novel species of the genus Marinobacter. Cells of the new isolates formed non-pigmented colonies and were motile by means of a single polar flagellum. Strains SD-14B(T) and SD-14C grew optimally at 35-37 degrees C, in 5 % NaCl and at pH 7.5-8.0. The organisms were aerobic, but reduced nitrate to nitrogen under anaerobic conditions. Acid was produced from only a few carbohydrates. Ubiquinone 9 was the major respiratory quinone. The major fatty acids of strains SD-14B(T) and SD-14C were C(16 : 0), C(18 : 1)omega9c, summed feature 3 (C(16 : 1)omega6c/C(16 : 1)omega7c) and C(12 : 0) 3-OH. The DNA G+C contents were 55.9 and 55.7 mol%, respectively. On the basis of the phylogenetic analyses and physiological and biochemical characteristics, it is proposed that strains SD-14B(T) and SD-14C represent a novel species of the genus Marinobacter, with the name Marinobacter salsuginis sp. nov. The type strain is strain SD-14B(T) (=DSM 18347(T)=LMG 23697(T)).