A bacterial consortium for treatment of Fat, Oil, and Grease (FOGs) in wastewater treatment plants

Fats, oils and greases (FOG), CER 190809, in wastewater create problems including the production of foul odours, the blockage of sewer lines and interference with the proper operation of sewage treatment works. Removal of FOG is thus critically important to ensure that wastewater is disposed of efficiently and economically. In this study, 232 bacterial strains were isolated and screened for the ability to degrade lipids from a specialized treatment plant, where a enhanced FOG degradation activity had been detected. Enrichment cultures on oily substrates, followed by isolation on selective media for lipase producing bacteria, and enzymatic methods were used to screen lipolytic microorganisms. In a second step, the lipolytic bacteria were analyzed using a colorimetric assay to detect the transesterification activity of para-nitrophenyl-palmitate. Four best performing lipid-degrading bacteria were identified by 16SrDNA sequencing, and investigated for application in treatment of lipids-contaminated wastewater, in Sequencing Batch Reactor (SBR) pilot plant. The FOG biodegradation efficiency was evaluated after 10 days using the gravimetric method for quantitative determination of total oily substances. The bacterial strains were Gram negative affiliated to Serratia, Aeromonas, Pedobacter genera. The Bacterial consortium was able to degrade 76% of FOG in 10 days treatment. The strains are be of great interest at industrial scale to increase the removal of FOG in wastewater treatment plants, and directly in the waste storage tanks at catering establishments, to reduce the polluting load before transfer to the disposal plan. The process of bioaugmentation using the FOG degrading consortium was recently patented (n°812021000056699

The impact of methanotrophic activity on methane emissions through the soils of geothermal areas

Methane plays an important role in the Earth’s atmospheric chemistry and radiative balance being the most important greenhouse gas after carbon dioxide. It has recently been established that geogenic gases contribute significantly to the natural CH4 flux to the atmosphere (Etiope et al., 2008). Volcanic/geothermal areas contribute to this flux, being the site of widespread diffuse degassing of endogenous gases (Chiodini et al., 2005). In such an environment soils are a source rather than a sink for atmospheric CH4 (Cardellini et al., 2003; Castaldi and Tedesco, 2005; D’Alessandro et al., 2009; 2011; 2013). Due to the fact that methane soil flux measurements are laboratory intensive, very few data have been collected until now in these areas. Preliminary studies (Etiope et al., 2007) estimated a total CH4 emission from European geothermal and volcanic systems in the range 4-16 kt a-1. This estimate was obtained indirectly from CO2 or H2O output data and from CO2/CH4 or H2O/CH4 values measured in the main gaseous manifestations. Such methods, although acceptable to obtain order-of-magnitude estimates, completely disregard possible methanotrophic activity within the soil. At the global scale, microbial oxidation in soils contributes for about 3-9% to the total removal of methane from the atmosphere. But the importance of methanotrophic organisms is even larger because they oxidise the greatest part of the methane produced in the soil and in the subsoil before its emission to the atmosphere. Environmental conditions in the soils of volcanic/geothermal areas (i.e. low oxygen content, high temperature and proton activity, etc.) have been considered inadequate for methanotrophic microrganisms. But recently, it has been demonstrated that methanotrophic consumption in soils occurs also under such harsh conditions due to the presence of acidophilic and thermophilic Verrucomicrobia. These organisms were found in Italy at the Solfatara di Pozzuoli (Pol et al., 2007), in New Zealand at Hell’s Gate (Dunfield et al., 2007) and in Kamchatka, Russia (Islam et al., 2008). Both the Italian and the Hellenic territories are geodynamically very active with many active volcanic and geothermal areas. Here we report on methane flux measurements made at Pantelleria (Italy) and at Sousaki and Nisyros (Greece). The total methane output of these three systems is about 10, 19 and 1 t a-1, respectively (D’Alessandro et al., 2009; 2011; 2013). The total emissions obtained from methane flux measurements are up to one order of magnitude lower than those obtained through indirect estimations. Clues of methanotrophic activity within the soils of these areas can be found in the CH4/CO2 ratio of the flux measurements which is always lower than that of the respective fumarolic manifestations, indicating a loss of CH4 during the travel of the gases towards earth’s surface. Furthermore laboratory methane consumption experiments made on soils collected at Pantelleria and Sousaki revealed, for most samples, CH4 consumption rates up to 9.50 μg h-1 and 0.52 μg h-1 respectively for each gram of soil (dry weight). Only few soil samples displayed no methane 2 consumption activity. Finally, microbiological and molecular investigations allowed us to identify the presence of methanotrophic bacteria belonging to the Verrucomicrobia and to the Alpha- and Gamma-Proteobacteria in the soils of the geothermal area of Favara Grande at Pantelleria. While the presence of the former was not unexpected due to the fact that they include acidophilic and thermophilic organisms that were previously found in other geothermal environments, the latter are generally considered not adapted to live in harsh geothermal environments. Their presence in the soils of Pantelleria could be explained by the fact that these soils do not have extremely low pH values (>5). Indeed thermotollerant methanotrophic Gamma-proteobacteria, have been previously found in the sediments of thermal springs in Kamchatka (Kizilova et al., 2012). Such species could find their niches in the shallowest part of the soils of Favara Grande were the temperatures are not so high and they thrive on the abundant upraising hydrothermal methane. References: Cardellini C., Chiodini G., Frondini F., Granieri D., Lewicki J., Peruzzi L., 2003. Accumulation chamber measurements of methane fluxes: application to volcanic–geothermal areas and landfills. Appl. Geochem. 18, 45–54. Castaldi S., Tedesco D., 2005. Methane production and consumption in an active volcanic environment of Southern Italy. Chemosphere 58, 131–139. Chiodini G., Granieri D., Avino R., Caliro S., Costa A., 2005. Carbon dioxide diffuse degassing and estimation of heat release from volcanic and hydrothermal systems. J. Geophys. Res. 110, B08204. D’Alessandro W., Bellomo S., Brusca L., Fiebig J., Longo M., Martelli M., Pecoraino G., Salerno F., 2009. Hydrothermal methane fluxes from the soil at Pantelleria island (Italy). J. Volcanol. Geotherm. Res. 187, 147–157. D’Alessandro W., Brusca L., Kyriakopoulos K., Martelli M., Michas G., Papadakis G., Salerno F., 2011. Diffuse hydrothermal methane output and evidence of methanotrophic activity within the soils at Sousaki (Greece). Geofluids 11, 97–107 D’Alessandro W., Gagliano A.L., Kyriakopoulos K., Parello F., 2013. Hydrothermal methane fluxes from the soil at Lakki plain (Nisyros island, Greece). Bull. Geol. Soc. Greece, vol. XLVII Proc. of the 13th International Congress, Chania, Sept. 2013 Dunfield P.F., Yuryev A., Senin P., Smirnova A.V., Stott M.B., Hou S., Ly B., Saw J.H., Zhou Z., Ren Y, Wang J., Mountain B.W., Crowe M.A., Weatherby T.M., Bodelier P.L.E., Liesack W., Feng L., Wang L., Alam M., 2007. Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia. Nature, 450, 879–882. Etiope G., Fridriksson T., Italiano F., Winiwarter W., Theloke J., 2007. Natural emissions of methane from geothermal and volcanic sources in Europe. J. Volcanol. Geotherm. Res. 165, 76–86. Etiope G., Lassey K.R., Klusman R.W., Boschi E., 2008. Reappraisal of the fossil methane budget and related emission from geologic sources. Geophys. Res. Lett. 35, L09307. Islam T., Jensen S., Reigstad L.J., Larsen Ø., Birkeland N.K., 2008. Methane oxidation at 55°C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum. Proc. Natl. Acad. Sci. 105, 300–304. Kizilova A.K., Dvoryanchikova E.N., Sukhacheva M.V., Kravchenko I.K., Gal’chenko V.F., 2012. Investigation of the communities of the Hot Springs of the Uzon Caldera, Kamchatka, by Molecular Ecological Techniques. Microbiology, 81, 606-613. Pol A., Heijmans K., Harhangi H.R., Tedesco D., Jetten M.S.M., Op den Camp H.J.M., 2007. Methanotrophy below pH 1 by a new Verrucomicrobia species. Nature, 450, 874–878

High diversity of methanotrophic bacteria in geothermal soils affected by high methane fluxes

Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas 25 times as potent as CO2. Recently, it has been demonstrated that volcanic/geothermal soils act as source, but also as biological filter for methane release to the atmosphere. For long time, volcanic/geothermal soils has been considered inhospitable for methanotrophic microorganisms, but new extremophile methanotrophs belonging to Verrucomicrobia were identified in three different areas (Pozzuoli, Italy; Hell’s Gate, New Zealand; Kamchatka, Russia), explaining anomalous behaviours in methane leakages of several geothermal/volcanic sites. Our aim was to increase the knowledge of the relationship between methane emissions from volcanic/geothermal areas and biological methane oxidation, by investigating a geothermal site of Pantelleria island (Italy). Pantelleria Island hosts a high enthalpy geothermal system characterized by high temperature, high CH4 and very low H2S fluxes. Such characteristics are reflected in potentially great supply of methane for methanotrophs and scarce presence of inhibitors of their activity (H2S and NH3) in the Pantelleria soils. Potential methanotrophic activity within these soils was already evidenced by the CH4/CO2 ratio of the flux measurements which was lower than that of the respective fumarolic manifestations indicating a loss of CH4 during the gas travel towards the earth’s surface. In this study laboratory incubation experiments using soils sampled at Favara Grande, the main hydrothermal area of Pantelleria, showed very high methane consumption rates (up to 9500 ng CH4 h1 g1). Furthermore, microbiological and culture-independent molecular analyses allowed to detect the presence of methanotrophs affiliated to Gamma- and Alpha-Proteobacteria and to the newly discovered acidothermophilic methanotrophs Verrucomicrobia. Culturable methanotrophic Alpha-proteobacteria of the genus Methylocystis were isolated by enrichment cultures. The isolates showed a wide range of tolerance to pH (3.5 – 8) and temperatures (18 – 45 C), and an average methane oxidation rate of 450 ppm/h. A larger diversity of proteobacterial and verrucomicrobial methanotrophs was detected by the amplification of the methane mono-oxygenase gene pmoA. This study demonstrates the coexistence of both the methanotrophic phyla Verrucomicrobia and Proteobacteria in the same geothermal site. The presence of proteobacterial methanotrophs was quite unexpected because they are generally considered not adapted to live in such harsh environments. Their presence at Favara Grande could be explained by not so low soil pH values (> 5) of this specific geothermal site and by the high methane availability. Such species could have found their niches in the shallowest part of the soils, were the temperatures are not so high, thriving on the abundant upraising methane. Understanding the ecology of methanotrophy in geothermal sites will increase our knowledge of their role in methane emissions to the atmosphere

The gut microbiota of the wood-feeding termite Reticulitermes lucifugus (Isoptera; Rhinotermitidae)

Termite gut is host to a complex microbial community consisting of prokaryotes, and in some cases flagellates, responsible for the degradation of lignocellulosic material. Here we report data concerning the analysis of the gut microbiota of Reticulitermes lucifugus (Rossi), a lower termite species that lives in underground environments and is widespread in Italy, where it causes damage to wood structures of historical and artistic monuments. A 16S rRNA gene clone library revealed that the R. lucifugus gut is colonized by members of five phyla in the domain Bacteria: Firmicutes (49 % of clones), Proteobacteria (24 %), Spirochaetes (14 %), the candidatus TG1 phylum (12 %), and Bacteroidetes (1 %). A collection of cellulolytic aerobic bacteria was isolated from the gut of R. lucifugus by enrichment cultures on different cellulose and lignocellulose substrates. Results showed that the largest amount of culturable cellulolytic bacteria of R. lucifugus belongs to Firmicutes in the genera Bacillus and Paenibacillus (67 %). These isolates are also able to grow on xylan and show the largest clear zone diameter in the Congo red test. Reticulitermes lucifugus hosts a diverse community of bacteria and could be considered an acceptable source of hydrolytic enzymes for biotechnological applications

Degradation of long-chain n-alkanes in soil microcosms by two actinobacteria

The ability of two recently isolated actinobacteria, that degrade medium and long chain n-alkanes in laboratory water medium, was investigated in soil microcosms using different standard soils that were artificially contaminated with n-alkanes of different length (C12- C20- C24- C30). The two strains, identified as Nocardia sp. SoB and Gordonia sp. SoCp, revealed a similar high HC degradation efficiency with an average of 75% alkane degraded after 28 days incubation. A selectivity of bacteria towards n-alkanes of different length was detected as well as a consistent effect of soil texture and other soil physical chemical characteristics on degradation. It was demonstrated the specific aptitude of these selected strains towards specific environmental conditions

Impact of water stress on plant water relations, growth and mycorhization of Acacia tortilis ssp. raddiana: Interactions of the stress severity and duration, and the AM fungal species.

Mycorrhizae are commonly reported to improve drought resistance of the host plant. Investigating factors controlling the plant-resistance strategy to water stress can however lead to a suitable management of mycorrhizal plants under drought. Here, we focused on the impact of interactions of the AM fungal species and water stress severity and duration on mycorrhizal Acacia tortilis ssp. raddiana in terms of plant growth, mycorhization and water relations. We used three Glomus species, which were G. mosseae, G. deserticola and G. intraradices. We applied three watering regimes that were maximum water holding non-stress regime, mild-severe water stress and severe water stress. Measurements were performed at four stage durations named pre-drought (one day before applying water stress), early-drought (30 days after applying water stress), mid-drought (60 days after applying water stress), and late-drought (90 days after applying water stress). Under maximum water holding regime, G. mosseae was more infective than G. deserticola and G. intraradices, but differences were not significantly different (P < 0.05). Predawn water potential was analogous for both mycorrhizal and control treatments whilst midday water potential was lower for control than for AMF treatments. Under mild and late drought, G. deserticola and G. intraradices were more infective than G. mosseae. Plants inoculated with G. intraradices had the highest predawn water potential and MWP. Mid-morning and midday stomatal conductance were similar and higher in seedlings inoculated with G. deserticola and G. intraradices. Under severe and late drought, infectivity was comparable and higher for both G. deserticola and G. intraradices. However, plants inoculated with G. intraradices displayed the highest growth, midday water potential, stomatal conductance and relative water content. Seedlings inoculated with G. mosseae and the controls had lost their leaves at -4 MPa water potential. Difference between Glomus species in the host response to water stress seemed to be related to the severity and duration of the stress. On the whole, inoculation of A. tortilis ssp. raddiana with G. intraradices resulted in increased plant growth and improved water status under severe and long-lasting drought, relative to G. deserticola and G. mosseae

Innovative ready to use carrier-bacteria devices for bioremediation of oil contaminated water

Bioremediation, that uses microorganisms to remove environmental pollutants, is the best way of restoring the environment due to its low cost and sustainability. Immobilization of microorganisms capable of degrading specific contaminants significantly promotes bioremediation processes. An innovative ready to use bioremediation system to clean up oil-contaminated water was developed immobilizing highly performant marine and soil HC degrading bacteria, on biodegradable oil-absorbing carriers. Two soil Actinobacteria (Gordonia sp. SoCg, Nocardia sp. SoB) and two marine Gammaproteobacteria (Alcanivorax sp. SK2, Oleibacter sp.5), were immobilized on biopolymeric membranes prepared by electrospinning (polylactic acid, PLA and polycaprolactone, PCL). These carriers are characterized by high uptake capacity, oil retention, buoyancy, durability, reusability and recoverability of the oil absorbed. The morphology of the carriers and microbial adhesion and proliferation were evaluated using scanning electron microscopy (SEM). A high capacity of adhesion and proliferation of bacterial cells was observed on membranes after 5 days. The bioremediation efficiency of the carrier-bacteria systems was tested on crude oil by GC-FID analysis and compared whit planktonic cells. The bacterial immobilization on PLA and PCL membranes was a promoting factor for biodegradation, increasing hydrocarbon removal up to 20%, in respect to planktonic cells. Biofilm-mediated bioremediation is a versatile tool to be developed for in situ and ex situ bioremediation of aquatic systems. Several applications can be designed to exploit both the high oil uptake capacity of the carriers, and the biodegradation potential of autochtonous microrganisms and/or of selected microorganisms that are immobilized on the carriers before exposure to the contaminated site

Innovative, ecofriendly biosorbent-biodegrading biofilms for bioremediation of oil- contaminated water

Immobilization of microorganisms capable of degrading specific contaminants significantly promotes bioremediation processes. In this study, innovative and ecofriendly biosorbent-biodegrading biofilms have been developed in order to remediate oil-contaminated water. This was achieved by immobilizing hydrocarbon-degrading gammaproteobacteria and actinobacteria on biodegradable oil-adsorbing carriers, based on polylactic acid and polycaprolactone electrospun membranes. High capacities for adhesion and proliferation of bacterial cells were observed by scanning electron microscopy. The bioremediation efficiency of the systems, tested on crude oil and quantified by gas chromatography, showed that immobilization increased hydrocarbon biodegradation by up to 23 % compared with free living bacteria. The resulting biosorbent biodegrading biofilms simultaneously adsorbed 100 % of spilled oil and biodegraded more than 66 % over 10 days, with limited environmental dispersion of cells. Biofilm-mediated bioremediation, using eco-friendly supports, is a low-cost, low-impact, versatile tool for bioremediation of aquatic systems

Methanotrophic activity and diversity of methanotrophs in volcanic-geothermal soils at Pantelleria island (Italy)

Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas twenty-five times as potent as CO2. Recently, it has been demonstrated that volcanic/geothermal soils are not only a source of methane, but also sites of methanotrophic activity. Methanotrophs are able to consume 10-40 Tg of CH4 a-1 and to trap more than 50% of the methane degassing through the soils. We report on methane microbial oxidation in the geothermally most active site of Pantelleria island (Italy), Favara Grande, whose total methane emission was previously estimated in about 2.5 Mg a-1 (t a-1). Laboratory incubation experiments with three top-soil samples from Favara Grande indicated methane consumption values up to 59.2 nmol g-1 soil d.w. h-1. One of the three sites, FAV2, where the highest oxidation rate was detected, was further analysed on a vertical soil profile and the maximum methane consumption was measured in the top-soil layer and values >6.23 nmol g-1 h-1 were still detected up to a depth of 13 cm. The highest consumption rate was measured at 37°C, but a still detectable consumption at 80°C (>1.25 nmol g -1 h-1) was recorded. The soil total DNAs extracted from the three samples were probed by PCR using standard proteobacterial primers and newly designed verrucomicrobial primers, targeting the unique methane monooxygenase gene pmoA; the presence of methanotrophs was detected in sites FAV2 and FAV3, but not in FAV1, where harsher chemical-physical conditions and negligible methane oxidation were detected. The pmoA gene libraries from the most active site FAV2 pointed out a high diversity of gammaproteobacterial methanotrophs, distantly related to Methylococcus/Methylothermus genera and the presence of the newly discovered acido-thermophilic methanotrophs Verrucomicrobia. Alphaproteobacteria of the genus Methylocystis were isolated from enrichment cultures, under a methane containing atmosphere at 37°C. The isolates grow at a pH range from 3.5 to 8, temperatures of 18 – 45 °C and consume 160 nmol of CH 4 h-1 ml-1 of culture. Soils from Favara Grande showed the largest diversity of methanotrophic bacteria until now detected in a geothermal soil. While methanotrophic Verrucomicrobia are reported to dominate highly acidic geothermal sites, our results suggest that slightly acidic soils, in high enthalpy geothermal systems, host a more diverse group of both culturable and uncultivated methanotrophs

The Root Mycobiota of Betula aetnensis Raf., an Endemic Tree Species Colonizing the Lavas of Mt. Etna (Italy)

Betula aetnensis is an endemic tree of high conservation value, which thrives on the nutrientpoor volcanic soils of Mount Etna. Since plant–microbe interactions could play a crucial role in plant growth, resource uptake, and resistance to abiotic stresses, we aimed to characterize the root and rhizosphere microbial communities. Individuals from natural habitat (NAT) and forest nursery (NURS) were surveyed through microscopy observations and molecular tools: bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA), fungal denaturing gradient gel electrophoresis (DGGE). B. aetnensis was found to be simultaneously colonized by arbuscular (AM), ectomycorrhizal (ECM), ericoid (ERM) fungi, and dark septate endophytes (DSE). A high diversity of the bacterial community was observed whilst the root fungal assemblage of NAT plants was richer than that of NURS. Root and rhizosphere fungal communities from NAT plants were characterized by Illumina MiSeq sequencing. Most of the identified sequences were affiliated to Helotiales, Pezizales, and Malasseziales. Ascomycota and Basidiomycota dominated roots and rhizosphere but differed in community structure and composition. ECM in the roots mainly belonged to Tylospora and Leccinum, while Rhizopogon was abundant in the rhizosphere. The Helotiales, including ERM (mostly Oidiodendron) and DSE (mostly Phialocephala), appeared the dominant component of the fungal community. B. aetnensis harbors an extraordinarily wide array of root-associated soil microorganisms, which are likely to be involved in the adaptation and resistance mechanisms to the extreme environmental conditions in volcano Etna. We argue that nursery-produced seedlings could lack the necessary microbiota for growth and development in natural conditions