Functional and structural responses of a halophilic consortium to oily sludge during biodegradation.

Biotreatment of oily sludge and the involved microbial communities, particularly in saline environments, have been rarely investigated. We enriched a halophilic bacterial consortium (OS-100) from petroleum refining oily sludge, which degraded almost 86% of the aliphatic hydrocarbon (C10-C30) fraction of the oily sludge within 7 days in the presence of 100 g/L NaCl. Two halophilic hydrocarbon-degrading bacteria related to the genera Chromohalobacter and Halomonas were isolated from the OS-100 consortium. Hydrocarbon degradation by the OS-100 consortium was relatively higher compared to the isolated bacteria, indicating potential synergistic interactions among the OS-100 community members. Exclusion of FeCl2, MgCl2, CaCl2, trace elements, and vitamins from the culture medium did not significantly affect the hydrocarbon degradation efficiency of the OS-100 consortium. To the contrary, hydrocarbon biodegradation dropped from 94.1 to 54.4% and 5% when the OS-100 consortium was deprived from phosphate and nitrogen sources in the culture medium, respectively. Quantitative PCR revealed that alkB gene expression increased up to the 3rd day of incubation with 11.277-fold, consistent with the observed increments in hydrocarbon degradation. Illumina-MiSeq sequencing of 16 S rRNA gene fragments revealed that the OS-100 consortium was mainly composed of the genera Halomonas, Idiomarina, Alcanivorax and Chromohalobacter. This community structure changed depending on the culturing conditions. However, remarkable changes in the community structure were not always associated with remarkable shifts in the hydrocarbonoclastic activity and vice versa. The results show that probably synergistic interactions between community members and different subpopulations of the OS-100 consortium contributed to salinity tolerance and hydrocarbon degradation.

Study of microbial communities and environmental parameters of seawater collected from three Tunisian fishing harbors in Kerkennah Islands: Statistical analysis of the temporal and spatial dynamics.

Surface seawater, collected from three fishing harbors during different seasons of the years 2015, 2016 and 2017, were assessed for physico-chemical analyses. Results showed that seawater was mainly polluted by hydrocarbons and some heavy metals. Microbial communities' composition and abundance in the studied harbors were performed using molecular approaches. SSCP analysis indicated the presence of Bacteria, Archaea and Eucarya, with dominance of the bacterial domain. Illumina Miseq analysis revealed that the majority of the sequences were affiliated with Bacteria whereas Archaea were detected at low relative abundance. The bacterial community, dominated by Proteobacteria, Bacteroidetes, Planctomycetes, Cyanobacteria, Firmicutes, Actinobacteria and Chloroflexi phyla, are known to be involved in a variety of biodegradation/biotransformation processes including hydrocarbons degradation and heavy metals resistance. The main objectives of this study are to assess, for the first time, the organic/inorganic pollution in surface seawater of Kerkennah Islands harbors, and to explore the potential of next generation marine microbiome monitoring to achieve the planning coastal managing strategies worldwide.

Biotreatment of oily sludge by a bacterial consortium: Effect of bioprocess conditions on biodegradation efficiency and bacterial community structure.

We studied the biodegradation of oily sludge generated by a petroleum plant in Bahrain by a bacterial consortium (termed as AK6) under different bioprocess conditions. Biodegradation of petroleum hydrocarbons in oily sludge (C11-C29) increased from 24% after two days to 99% after 9 days of incubation in cultures containing 5% (w/v) of oily sludge at 40°C. When the nitrogen source was excluded from the batch cultures, hydrocarbon biodegradation dropped to 45% within 7 days. The hydrocarbon biodegradation decreased also by increasing the salinity to 3% and the temperature above 40°C. AK6 tolerated up to 50% (w/v) oily sludge and degraded 60% of the dichloromethane-extractable oil fraction. Illumina-MiSeq analyses revealed that the AK6 consortium was mainly composed of Gammaproteobacteria (ca. 98% of total sequences), with most sequences belonging to Klebsiella (77.6% of total sequences), Enterobacter (16.7%) and Salmonella (5%). Prominent shifts in the bacterial composition of the consortium were observed when the temperature and initial sludge concentration increased, and the nitrogen source was excluded, favoring sequences belonging to Pseudomonas and Stenotrophomonas. The AK6 consortium is endowed with a strong oily sludge tolerance and biodegradation capability under different bioprocess conditions, where Pseudomonas spp. appear to be crucial for hydrocarbon biodegradation.

Lipopeptides production by a newly Halomonas venusta strain: Characterization and biotechnological properties.

A halotolerant marine strain PHKT of Halomonas venusta was isolated from contaminated seawater as an efficient biosurfactant producer candidate, on low-value substrate (glycerol). The produced biosurfactants (Bios-PHKT) were characterized as lipopeptides molecules, belonging to surfactin and pumilacidin families, by using Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FT-IR) and Tandem Mass Spectrometry (MALDI-TOF/MS-MS). Bios-PHKT has a critical micelle concentration (CMC) of 125 mg/L, and showed a high steadiness against a wide spectrum of salinity (0-120 g/L NaCl), temperature (4-121 °C) and pH (2-12), supporting its powerful tensioactive properties under various environmental conditions. Likewise, the cytotoxic test revealed that the biosurfactant Bios-PHKT, at concentrations lower than 125 µg/mL, was not cytotoxic for human HEK-293 cells since the cell survival is over than 80%. Furthermore, Bios-PHKT lipopeptides showed excellent anti-adhesive and anti-biofilm activities, being able to avoid and disrupt the biofilm formation by certain pathogenic microorganisms. In addition, the biosurfactant Bios-PHKT showed a remarkable anti-proliferative activity towards tumor B16 melanoma cell line. Besides, Bios-PHKT exhibited an excellent in vitro and in vivo wound healing process. In light of these promising findings, Bios-PHKT could be successfully used in different biotechnological applications.

Investigation of halotolerant marine Staphylococcus sp. CO100, as a promising hydrocarbon-degrading and biosurfactant-producing bacterium, under saline conditions.

A halotolerant strain CO100 of Staphylococcus sp. was isolated from contaminated sediments taken from the fishing harbour of Sfax, Tunisia, as an efficient hydrocarbonoclastic candidate. Strain CO100 exhibited a high capacity to break down almost 72% of the aliphatic hydrocarbons contained in crude oil (1%, v/v), used as the sole carbon and energy source, after 20 days of culture, at 100 g/l NaCl, 37 °C and 180 rpm. The isolate CO100 displayed also its ability to grow on phenanthrene, fluoranthene and pyrene (100 mg/l), at 100 g/l NaCl. Moreover, the isolate CO100 showed a notable aptitude to synthesize an efficient tensioactive agent namely BS-CO100, on low-value substrates including residual frying oil and expired milk powder, thus reducing the high cost of biosurfactant production. The ESI/MS analysis designated that BS-CO100 belonged to lipopeptide class, in particular lichenysin and iturine members. Critical micelle concentrations of BS-CO100 were varying between 65 and 750 mg/l, depending on of the purity of the biosurfactant and the used carbon sources. BS-CO100 showed a high steadiness against a wide spectrum of pH (4.3-12), temperature (4-121 °C) and salinity (0-300 g/l NaCl), supporting its powerful tensioactive properties under various environmental conditions. Likewise, BS-CO100 exhibited no cytotoxic effect toward human HEK293 cells, at concentrations within 125 and 1000 µg/ml. Furthermore, the biosurfactant BS-CO100 exhibited remarkable anti-adhesive and anti-biofilm activities, being able to avoid and disrupt the biofilm formation by certain pathogenic microorganisms. In addition, BS-CO100 was found to have more potential to remove hydrocarbons from contaminated soils, compared to some chemical surfactants. In light of these promising findings, strain CO100, as well as its biosurfactant, could be successfully used in different biotechnological applications including the bioremediation of oil-polluted areas, even under saline conditions.

Isolation and characterization of a newly naphthalene-degrading Halomonas pacifica, strain Cnaph3: biodegradation and biosurfactant production studies.

A newly marine Halomonas pacifica strain Cnaph3 was isolated, as a naphthalene degrader and biosurfactant producer, from contaminated seawater collected in Ataya's fishing harbor, located in Kerkennah Islands, Tunisia. Chromatography flame ionization detector analysis revealed that 98.8% of naphthalene (200 mg/L) was degraded after 7 days of incubation, at 30 g/L NaCl and 37 °C. Strain Cnaph3 showed also a noticeable capacity to grow on a wide range of aliphatic, aromatic, and complex hydrocarbons. Interestingly, strain Cnaph3 showed a significant potential to produce biosurfactants in the presence of all tested substrates, particularly on glycerol (1%, v/v). Electrospray ionization analysis of the biosurfactant, designated Bios-Cnaph3, suggested a lipopeptide composition. The critical micelle concentration of Bios-Cnaph3 was about 500 mg/L. At this concentration, the surface tension of the water was reduced to 27.6 mN/m. Furthermore, Bios-Cnaph3 displayed interesting stabilities over a wide range of temperatures (4-105 °C), salinities (0-100 g/L NaCl), and pH (2.2-12.5). In addition, it showed promising capacities to remove used motor oil from contaminated soils. The biodegradation and biosurfactant-production potential of the Halomonas sp. strain Cnaph3 would present this strain as a favorite agent for bioremediation of hydrocarbon-contaminated sites under saline conditions.

Production, characterization and biotechnological potential of lipopeptide biosurfactants from a novel marine Bacillus stratosphericus strain FLU5.

This work aimed at studying the potential of a new hydrocarbonoclastic marine bacterium, Bacillus stratosphericus FLU5, to produce an efficient surface-active agent BS-FLU5. Biosurfactant production was examined on different carbon sources; using the surface tension measurement and the oil displacement test. Strain FLU5 showed its capacity to produce biosurfactants from all tested substrates, in particular the residual frying oil, which is a cheap renewable carbon source alternative, thus minimizing the high cost of producing those surfactants. MALDI-TOF MS/MS analysis confirmed the presence of lipopeptides, which are identified as members of surfactin and pumilacidin series. The critical micelle concentration (CMC) of the purified lipopeptides produced by strain FLU5 was 50 mg/l. At this concentration, the surface tension of the water was reduced from 72 to 28 mN/m. Furthermore, the crude lipopeptides showed an interesting stability against a broad range of pH, temperature and salinity. In addition, the application of BS-FLU5 in oil recovery from hydrocarbons-contaminated soil (used motor oil) showed that it was more effective on the hydrocarbon-remobilization than some tested synthetic surfactants. Interestingly, the biosurfactant BS-FLU5 showed a negligible cytotoxic effect against the mammalian cells HEK293. These results highlight the applicability of the lipopeptides BS-FLU5 in different fields, especially in environmental remediation processes.

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.

Preliminary characterization of biosurfactant produced by a PAH-degrading Paenibacillus sp. under thermophilic conditions.

The capacities of a biosurfactant producing and polycyclic aromatic hydrocarbon (PAH) utilizing bacterium, namely, strain 1C, isolated from an Algerian contaminated soil, were investigated. Strain 1C belonged to the Paenibacillus genus and was closely related to the specie Paenibacillus popilliae, with 16S rRNA gene sequence similarity of 98.4 %. It was able to produce biosurfactant using olive oil as substrate. The biosurfactant production was shown by surface tension (32.6 mN/m) after 24 h of incubation at 45 °C and 150 rpm. The biosurfactant(s) retained its properties during exposure to elevated temperatures (70 °C), relatively high salinity (20 % NaCl), and a wide range of pH values (2-10). The infrared spectroscopy (FTIR) revealed that its chemical structure belonged to lipopeptide class. The critical micelle concentration (CMC) of this biosurfactant was about 0.5 g/l with 29.4 mN/m. In addition, the surface active compound(s) produced by strain 1C enhanced PAH solubility and showed a significant antimicrobial activity against pathogens. In addition to its biosurfactant production, strain 1C was shown to be able to utilize PAHs as the sole carbon and energy sources. Strain 1C as hydrocarbonoclastic bacteria and its interesting surface active agent may be used for cleaning the environments polluted with polyaromatic hydrocarbons.

Biodegradation of fluoranthene by a newly isolated strain of Bacillus stratosphericus from Mediterranean seawater of the Sfax fishing harbour, Tunisia.

A physico-chemical characterization of seawater taken from the fishing harbour of Sfax, Tunisia, revealed a contamination by organic and inorganic micropollutants. An aerobic marine halotolerant Bacillus stratosphericus strain FLU5 was isolated after enrichment on fluoranthene, a persistent and toxic polycyclic aromatic hydrocarbon (PAH). GC-MS analyses showed that strain FLU5 was capable of degrading almost 45 % of fluoranthene (100 mg l(-1)), without yeast extract added, after 30 days of incubation at 30 g l(-1) NaCl and 37 °C. In addition, the isolate FLU5 showed a remarkable capacity to grow on a wide range of aliphatic, aromatic and complex hydrocarbons. This strain could also synthesize a biosurfactant which was capable of reducing the surface tension of the cell-free medium, during the growth on fluoranthene. The biodegradative abilities of PAHs are promising and can be used to perform the bioremediation strategies of seawaters and marine sediments contaminated by hydrocarbons.