Marine copepod culture as a potential source of bioplastic-degrading microbiome: The case of poly(butylene succinate-co-adipate).

The poly(butylene succinate-co-adipate) (PBSA) is emerging as environmentally sustainable polyester for applications in marine environment. In this work the capacity of microbiome associated with marine plankton culture to degrade PBSA, was tested. A taxonomic and functional characterization of the microbiome associated with the copepod Acartia tonsa, reared in controlled conditions, was analysed by 16S rDNA metabarcoding, in newly-formed adult stages and after 7 d of incubation. A predictive functional metagenomic profile was inferred for hydrolytic activities involved in bioplastic degradation with a particular focus on PBSA. The copepod-microbiome was also characterized in newly-formed carcasses of A. tonsa, and after 7 and 33 d of incubation in the plankton culture medium. Copepod-microbiome showed hydrolytic activities at all developmental stages of the alive copepods and their carcasses, however, the evenness of the hydrolytic bacterial community significantly increased with the time of incubation in carcasses. Microbial genera, never described in association with copepods: Devosia, Kordia, Lentibacter, Methylotenera, Rheinheimera, Marinagarivorans, Paraglaciecola, Pseudophaeobacter, Gaiella, Streptomyces and Kribbella sps., were retrieved. Kribbella sp. showed carboxylesterase activity and Streptomyces sp. showed carboxylesterase, triacylglycerol lipase and cutinase activities, that might be involved in PBSA degradation. A culturomic approach, adopted to isolate bacterial specimen from carcasses, led to the isolation of the bacterial strain, Vibrio sp. 01 tested for the capacity to promote the hydrolysis of the ester bonds. Granules of PBSA, incubated 82 d at 20 °C with Vibrio sp. 01, were characterized by scanning electron microscopy, infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry, showing fractures compared to the control sample, and hydrolysis of ester bonds. These preliminary results are encouraging for further investigation on the ability of the microbiome associated with plankton to biodegrade polyesters, such as PBSA, and increasing knowledge on microorganisms involved in bioplastic degradation in marine environment.

A New Ciboria sp. for Soil Mycoremediation and the Bacterial Contribution to the Depletion of Total Petroleum Hydrocarbons.

A Ciboria sp. strain (Phylum Ascomycota) was isolated from hydrocarbon-polluted soil of an abandoned oil refinery in Italy. The strain was able to utilize diesel oil as a sole carbon source for growth. Laboratory-scale experiments were designed to evaluate the use of this fungal strain for treatment of the polluted soil. The concentration of total petroleum hydrocarbons (TPH) in the soil was 8,538 mg/kg. Mesocosms containing the contaminated soil were inoculated with the fungal strain at 1 or 7%, on a fresh weight base ratio. After 90 days of incubation, the depletion of TPH contamination was of 78% with the 1% inoculant, and 99% with the 7% inoculant. 16S rDNA and ITS metabarcoding of the bacterial and fungal communities was performed in order to evaluate the potential synergism between fungi and bacteria in the bioremediation process. The functional metagenomic prediction indicated Arthrobacter, Dietzia, Brachybacerium, Brevibacterium, Gordonia, Leucobacter, Lysobacter, and Agrobacterium spp. as generalist saprophytes, essential for the onset of hydrocarbonoclastic specialist bacterial species, identified as Streptomyces, Nocardoides, Pseudonocardia, Solirubrobacter, Parvibaculum, Rhodanobacter, Luteiomonas, Planomicrobium, and Bacillus spp., involved in the TPH depletion. The fungal metabolism accelerated the onset of specialist over generalist bacteria. The capacity of the Ciboria sp. to deplete TPH in the soil in treatment was also ascertained.

Degradation of BTEX mixture by a new Pseudomonas putida strain: role of the quorum sensing in the modulation of the upper BTEX oxidative pathway.

A new Pseudomonas putida strain (AQ8) was isolated from a decommissioned oil refinery's soil in Italy and characterized for its ability to degrade BTEX. The draft genome of the new strain was sequenced and annotated for genes that encode enzymes putatively involved in BTEX degradation and quorum sensing. The strain was transformed with a plasmid expressing lactonase, which cleaves the autoinducer quorum sensing signal molecule, the acyl-homoserine lactone, to obtain a quorum sensing minus strain. P. putida AQ8 depleted the 40% on average of all the components of the initial BTEX concentration in 36 h. The quorum sensing minus strain, in the same time interval, depleted only the 10% of the initial BTEX concentration. The role of quorum sensing in regulating the expression of the annotated benzene/toluene dioxygenase gene (benzA) and biphenyl/toluene/benzene dioxygenase (bphA) genes, which are involved in BTEX degradation, was studied by quantitative RT-real-time quantitative (q)PCR analysis. The qPCR data showed decreased levels of expression of the benzA and bphA genes in the quorum sensing minus strain. Our results showed, for the first time, quorum sensing modulation of the level of transcription of dioxygenase genes in the upper BTEX oxidation pathway.

The role of cosubstrate and mixing on fungal biofilm efficiency in the removal of tannins.

Tannins are polyphenolic compounds produced by plants and they are used in industrial vegetable tanning of leather. Tannins represent one of the low biodegradability substances in tannery wastewaters with high recalcitrant soluble chemical oxygen demand, furthermore high concentration of tannins can inhibit biological treatment. In the present study, four novel rotating submerged packed bed reactors were inoculated with a selected fungal strain to reach a biological degradation of tannins in non-sterile conditions. The selected fungal strain, Aspergillus tubingensis MUT 990, was immobilised in polyurethane foam cubes carriers and inserted inside a submerged rotating cage reactors. The reactors were feed with a solution composed of four tannins: Quebracho (Schinopsis spp.), Wattle (Mimosa spp.), Chestnut (Castanea spp.) and Tara (Caesalpinia spp.). Four reactors with a volume of 4 L each were used, the co-substrate was pure malt extract, the hydraulic retention time was 24 h and the pH setpoint was 5.5. The reactors configuration was chosen to allow the study of the effect of rotation and the co-substrate addition on tannins removal. The experiment lasted two months and it was achieved 80% of chemical oxygen demand and up to 90% dissolved organic carbon removal, furthermore it was detected an important tannase activity.

Hydrocarbonoclastic Ascomycetes to enhance co-composting of total petroleum hydrocarbon (TPH) contaminated dredged sediments and lignocellulosic matrices.

Four new Ascomycete fungi capable of degrading diesel oil were isolated from sediments of a river estuary mainly contaminated by shipyard fuels or diesel oil. The isolates were identified as species of Lambertella, Penicillium, Clonostachys, and Mucor. The fungal candidates degraded and adsorbed the diesel oil in suspension cultures. The Lambertella sp. isolate displayed the highest percentages of oxidation of diesel oil and was characterised by the capacity to utilise the latter as a sole carbon source. This isolate showed extracellular laccase and Mn-peroxidase activities in the presence of diesel oil. It was tested for capacity to accelerate the process of decontamination of total petroleum hydrocarbon contaminated sediments, co-composted with lignocellulosic residues and was able to promote the degradation of 47.6% of the TPH contamination (54,074 ± 321 mg TPH/Kg of sediment) after two months of incubation. The response of the bacterial community during the degradation process was analysed by 16S rRNA gene meta-barcoding.

PCB in the environment: bio-based processes for soil decontamination and management of waste from the industrial production of Pleurotus ostreatus.

Polychlorinated biphenyls (PCBs) are hazardous soil contaminants for which a bio-based technology for their recovery is essential. The objective of this study was to validate the exploitation of spent mushroom substrate (SMS), a low or null cost organic waste derived from the industrial production of P. ostreatus, as bulking agent in a dynamic biopile pilot plant. The SMS shows potential oxidative capacity towards recalcitrant compounds. The aim was consistent with the design of a process of oxidation of highly chlorinated PCBs, which is independent from their reductive dehalogenation. Feasibility was verified at a mesocosm scale and validated at pilot scale in a dynamic biopile pilot plant treating ten tons of a historically contaminated soil (9.28±0.08mg PCB/kg soil dry weight). Mixing of the SMS with the soil was required for the depletion of the contaminants. At the pilot scale, after eight months of incubation, 94.1% depletion was recorded. A positive correlation between Actinobacteria and Firmicutes active metabolism, soil laccase activity and PCB removal was observed. The SMS was found to be exploitable as a versatile low cost organic substrate capable of activating processes for the oxidation of highly chlorinated PCBs. Moreover, its exploitation as bulking agent in biopiles is a valuable management strategy for the re-utilisation of an organic waste deriving from the industrial cultivation of edible mushrooms.

Isolation and characterization of a hydrocarbonoclastic bacterial enrichment from total petroleum hydrocarbon contaminated sediments: potential candidates for bioaugmentation in bio-based processes.

Seven hydrocarbonoclastic new bacterial isolates were isolated from dredged sediments of a river estuary in Italy. The sediments were contaminated by shipyard activities since decades, mainly ascribable to the exploitation of diesel oil as the fuel for recreational and commercial navigation of watercrafts. The bacterial isolates were able to utilize diesel oil as sole carbon source. Their metabolic capacities were evaluated by GC-MS analysis, with reference to the depletion of both the normal and branched alkanes, the nC18 fatty acid methyl ester and the unresolved complex mixture of organic compounds. They were taxonomically identified as different species of Stenotrophomonas and Pseudomonas spp. by the combination of amplified ribosomal DNA restriction analysis (ARDRA) and repetitive sequence-based PCR (REP-PCR) analysis. The metabolic activities of interest were analyzed both in relation to the single bacterial strains and to the combination of the latter as a multibacterial species system. After 6 days of incubation in mineral medium with diesel oil as sole carbon source, the Stenotrophomonas sp. M1 strain depleted 43-46 % of Cn-alkane from C28 up to C30, 70 % of the nC18 fatty acid methyl ester and the 46 % of the unresolved complex mixture of organic compounds. On the other hand, the Pseudomonas sp. NM1 strain depleted the 76 % of the nC18 fatty acid methyl ester, the 50 % of the unresolved complex mixture of organic compounds. The bacterial multispecies system was able to completely deplete Cn-alkane from C28 up to C30 and to deplete the 95 % of the unresolved complex mixture of organic compounds. The isolates, either as single strains and as a bacterial multispecies system, were proposed as candidates for bioaugmentation in bio-based processes for the decontamination of dredged sediments.

Polycyclic aromatic hydrocarbon-contaminated soils: bioaugmentation of autochthonous bacteria and toxicological assessment of the bioremediation process by means of Vicia faba L.

Two bacterial strains, Achromobacter sp. (ACH01) and Sphingomonas sp. (SPH01), were isolated from a heavily polycyclic aromatic hydrocarbon (PAH)-contaminated soil (5431.3 ± 102.3 ppm) for their capacity to use a mixture of anthracene, pyrene, phenanthrene and fluorene as sole carbon sources for growth and for the capacity to produce biosurfactants. The two strains were exploited for bioaugmentation in a biopile pilot plant to increase the bioavailability and the degradation of the residual PAH contamination (99.5 ± 7.1 ppm) reached after 9 months of treatment. The denaturing gel gradient electrophoresis (DGGE) profile of the microbial ecology of the soil during the experimentation showed that the bioaugmentation approach was successful in terms of permanence of the two strains in the soil in treatment. The bioaugmentation of the two bacterial isolates positively correlated with the PAH depletion that reached 7.9 ± 2 ppm value in 2 months of treatment. The PAH depletion was assessed by the loss of the phyto-genotoxicity of soil elutriates on the model plant Vicia faba L., toxicological assessment adopted also to determine the minimum length of the decontamination process for obtaining both the depletion of the PAH contamination and the detoxification of the soil at the end of the process. The intermediate phases of the bioremediation process were the most significant in terms of toxicity, inducing genotoxic effects and selective DNA fragmentation in the stem cell niche of the root tip. The selective DNA fragmentation can be related to the selective induction of cell death of mutant stem cells that can compromise offsprings.

Phytomediated biostimulation of the autochthonous bacterial community for the acceleration of the depletion of polycyclic aromatic hydrocarbons in contaminated sediments.

Polycyclic aromatic hydrocarbons (PAHs) are a large group of organic contaminants causing hazards to organisms including humans. The objective of the study was to validate the vegetation of dredged sediments with Phragmites australis as an exploitable biostimulation approach to accelerate the depletion of PAHs in nitrogen spiked sediments. Vegetation with Phragmites australis resulted in being an efficient biostimulation approach for the depletion of an aged PAHs contamination (229.67 ± 15.56 µg PAHs/g dry weight of sediment) in dredged sediments. Phragmites australis accelerated the oxidation of the PAHs by rhizodegradation. The phytobased approach resulted in 58.47% of PAHs depletion. The effects of the treatment have been analyzed in terms of both contaminant depletion and changes in relative abundance of the metabolically active Gram positive and Gram negative PAHs degraders. The metabolically active degraders were quantified both in the sediments and in the root endospheric microbial community. Quantitative real-time PCR reactions have been performed on the retrotranscribed transcripts encoding the Gram positive and Gram negative large α subunit (RHDα) of the aromatic ring hydroxylating dioxygenases. The Gram positive degraders resulted in being selectively favored by vegetation with Phragmites australis and mandatory for the depletion of the six ring condensed indeno[1,2,3-cd]pyrene and benzo[g,h,i]perylene.