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BACKGROUND 1,1,2,2-tetrachloroethane (TeCA) has been generally considered as non-biodegradable under aerobic conditions, while its complete biodegradation was reported with microbial consortia growing anaerobically. This study describes TeCA aerobic co-metabolic degradation by the propanotroph Rhodococcus aetherivorans strain TPA isolated from a TeCA-degrading consortium. RESULTS R. aetherivorans TPA was able to grow on aliphatic hydrocarbons from propane to pentane and on gaseous n-alkane metabolic intermediates. The Michaelis–Menten model allowed a satisfactory fit of the TPA propane utilization rates under resting cell conditions, while the TeCA degradation rates were successfully interpolated with Andrew's inhibition model. A significant propane–TeCA mutual inhibition was observed, although the results did not allow distinguishing between competitive and non-competitive inhibition. Among different bioreactor options for the on-site bioremediation of TeCA-contaminated groundwater, a single suspended-cell continuous stirred-tank reactor (CSTR) appeared to be the optimal one. CONCLUSIONS This study provides for the first time the kinetic and microbiological characterization of a bacterial strain capable of degrading TeCA under aerobic conditions.

A genome analysis of Pseudomonas pseudoalcaligenes KF707, a PCBs degrader and metal-resistant soil microorganism, revealed the presence of two novel gene clusters named che2 and che3, which were predicted to be involved in chemotaxis-like pathways, in addition to a che1 gene cluster. We herein report that the histidine kinase coding genes, cheA2 and cheA3, have no role in swimming or chemotaxis in P. pseudoalcaligenes KF707, in contrast to cheA1. However, the cheA1 and cheA2 genes were both necessary for cell swarming, whereas the cheA3 gene product had a negative effect on the optimal swarming phenotype of KF707 cells.

“Terre Calde di Medolla” (TCM) (literally, “Hot Lands of Medolla”) refers to a farming area in Italy with anomalously high temperatures and diffuse emissions of biogenic CO2, which has been linked to CH4 oxidation processes from a depth of 0.7 m to the surface. We herein assessed the composition of the total bacterial community and diversity of methane-oxidizing bacteria (MOB) in soil samples collected at a depth at which the peak temperature was detected (0.6 m). Cultivation-independent methods were used, such as: i) a clone library analysis of the 16S rRNA gene and pmoA (coding for the α-subunit of the particulate methane monooxygenase) gene, and ii) Terminal Restriction Fragment Length Polymorphism (T-RFLP) fingerprinting. The 16S rRNA gene analysis assessed the predominance of Actinobacteria, Acidobacteria, Proteobacteria, and Bacillus in TCM samples collected at a depth of 0.6 m along with the presence of methanotrophs (Methylocaldum and Methylobacter) and methylotrophs (Methylobacillus). The phylogenetic analysis of pmoA sequences showed the presence of MOB affiliated with Methylomonas, Methylocystis, Methylococcus, and Methylocaldum in addition to as yet uncultivated and uncharacterized methanotrophs. Jaccard’s analysis of T-RFLP profiles at different ground depths revealed a similar MOB composition in soil samples at depths of 0.6 m and 0.7 m, while this similarity was weaker between these samples and those taken at a depth of 2.5 m, in which the genus Methylocaldum was absent. These results correlate the anomalously high temperatures of the farming area of “Terre Calde di Medolla” with the presence of microbial methane-oxidizing bacteria.

In a healthy host, a balance exists between members of the microbiota, such that potential pathogenic and non-pathogenic organisms can be found in apparent harmony. During infection, this balance can become disturbed, leading to often dramatic changes in the composition of the microbiota. For most bacterial infections, nonspecific antibiotics are used, killing the non- pathogenic members of the microbiota as well as the pathogens and leading to a substantial delay in the restoration of a healthy microbiota. Lactobacillus species dominate the vaginal niche of healthy reproductive age women and are involved in the homeostasis maintenance by preventing the overgrowth of urogenital pathogens. Lactobacilli play key protective roles through different mechanisms such as production of various antimicrobial compounds, co-aggregation, competitive exclusion, immunomodulation and release of biosurfactants (BS). Aim of the study was to investigate the ability of vaginal lactobacilli to produce Biosurfactants (BS), as well as to characterize BS in terms of anti-adhesive and anti-biofilm activities against some representative vaginal pathogens. Production of BS was evaluated by the oil displacement test, which is an indirect, sensitive and simple method for the measurement of molecules’ surface activity. Several lactobacilli strains produced significant amount of biosurfactants: BS produced by L. gasseri BC9 showed the best surface tension at both the highest (10 mg/ml) and the lowest (1.3 mg/ml) concentration. Anti- adhesive and anti-biofilm activities of BS produced by L. gasseri BC9 was evaluated against strains of Staphylococcus aureus and Escherichia coli, respectively. In conclusion, BS isolated from L. gasseri BC9 strain showed antibacterial properties against S. aureus. BS from BC9 strain also displayed anti-adhesive and anti-biofilm abilities mainly against S. aureus. Together, these capabilities may open up possibilities for BS as an alternative therapeutic approach for the prevention and/or treatment of Gram positive hospital-acquired infections.

Methane-oxidizing bacteria (methanotrophs) have a ubiquitous distribution in the environment and play an important role in global climate warming by lowering methane emission into the atmosphere. “Terre Calde di Medolla” (literally, “Warm Earths of Medolla”) refers to a farming area, located near the town of Modena (Emilia-Romagna region, northern Italy), which has always been known for the relatively high temperatures of the soil. After the 2012 Emilia earthquake that affected this area, soil temperatures of 44°C were revealed (20–25°C above the local background value) together with diffuse soil fluxes of methane (CH4) and minor, though significant, of carbon dioxide (CO2). Ground heating and gas seepage appeared to be spatially correlated, thus suggesting a close relationship between the two phenomena.The aim of this work was to characterize the methanotrophic microbial community present in soil samples collected from this area using a cultivation-independent approach. We performed clone library analysis and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the 16S rDNA and pmoA gene (coding for the alpha-subunit of the particulate methane monooxygenase). Results from 16S rDNA analysis showed that methanotrophic bacteria represented around 10% of the entire microbial community. In particular, bacteria belonging to Acidobacteria and Actinobacteria phyla were detected as well as members of Ammonifex, Sphingomonas and Bacillus genera. Through the analysis of pmoA gene, Methylococcus, Methylocaldum and Methylomonas were found to be the dominant genera (around 90%) of the methanotrophic community under analysis. In addition to these Gammaproteobacteria, Methylocystis spp. were detected which belong to Alphaproteobacteria class. Further studies will define the correlation between the composition and the role of the methanotrophic microbial community and the peculiar characteristics of “Terre Calde di Medolla” soil.

Naphthenic acids (NAs) are an important group of organic pollutants comprised predominantly of saturated aliphatic and alicyclic carboxylic acids. Although these compounds are toxic, recalcitrant, and persistent in oil sands deposits, we are just learning the diversity of microorganisms involved in NAs-degradation and the mechanisms by which NAs are biodegraded. Here, we described the ability of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 to utilize two model naphthenic acids (NAs) i.e. cyclohexancarboxylic acid (CHCA) and cyclopentanecarboxylic acid (CPCA) as sole carbon and energy source. BCP1 strain degraded both the NAs with higher rates compared to R7; moreover, CHCA was shown to be metabolized faster than CPCA by both the Rhodococcus spp. Furthermore, genes coding for enzymes putatively involved in β-oxidation metabolism were identified in BCP1 genome. RT-PCR experiments showed that all these genes are induced by the growth of BCP1 on either CPCA or CHCA. These results suggest their involvement in β-oxidation metabolism of NAs by R. sp. BCP1.

BACKGROUND. Bio-hydrogen production from organic residues is an attractive process that combines energy generation with waste treatment. This work represents the continuation of a previous study of biological H2 production from food industry wastes (Cappelleti et al., J. Chem. Technol. Biotechnol. 87:1291-1301), where the comparison of four thermophilic Thermotoga strains and four carriers for biomass immobilization led to the identification of T. neapolitana as the best-performing strain and of a porous ceramic material named Biomax as the best-performing support for biofilm formation. AIMS. The goals of this work were: a) to develop and calibrate a kinetic model of H2 production from glucose, molasses and cheese whey by suspended and attached cells of T. neapolitana, and b) to set-up and optimize a continuous-flow process of H2 production from cheese whey by T. neapolitana in a 20-L stirred reactor. METHODS. The experimental H2 rates used to develop the kinetic model were obtained from suspended- and attached-cell batch tests conducted in 120-mL bioreactors, exposed to different initial concentrations of glucose, molasses or cheese whey (0.1-95 g L-1, as total sugars). The process was scaled-up in a 20-L fermentor, fed only with cheese whey in consideration of the significantly higher production of this waste in Northern Italy, in comparison with molasses. The fermentor was operated initially with suspended-cells, and subsequently with attached-cells. To immobilize T. neapolitana in the fermentor, 5 L of the previously selected carrier were introduced in pockets made with steel net and fixed to the top and bottom of the vessel. RESULTS. The H2 specific production rates obtained with suspended and attached cell tests of T. neapolitana are shown in Figure 1, together with the best fitting simulations performed with the Haldane substrate inhibition model. The corresponding values of the kinetic parameters are reported in Table 1. For all the substrates studied, a marked substrate inhibition was noticed. The attached-cell tests resulted in higher rates than the suspended-cell ones, in particular in the case of glucose and molasses. Further tests indicated the absence of oxygen inhibition (studied range: 0-1% in the headspace, or 0-0.21 mgdissolved O2 L-1) and of product (H2) inhibition (studied range: 0-60% in the headspace, or 0-0.73 mgdissolved H2 L-1). The batch tests of H2 production from cheese whey conducted in the 20-L fermentor led to a productivity of 6.8 mmolH2 L-1 day-1 with suspended cells, and 22.5 mmolH2 L-1 day-1 with attached cells. The fermentor was also operated with attached cells in continuous-flow mode, with hydraulic retention times (HRT) in the 0.8-2.9 day range. The resulting H2 productivity showed an increasing trend with decreasing HRT, with a maximum value of 125 mmolH2 L-1 day-1. CONCLUSIONS. The calibration of a model of thermophilic H2 production from different wastes and the successful scale-up of the process of cheese whey conversion into H2 in a continuous-flow 20-L bioreactor pose the basis for a model-based optimization of the attached-cell process, and for its further scale-up to a pilot plant.

This work is aimed at evaluating the feasibility of a cost-effective process of biological H2 production from food industry wastes under thermophilic conditions. The H2-producing performances of 4 Thermotoga strains (T. neapolitana, T. petrophila, T. naphtophila, T. maritima) were compared at 77 °C by means of tests conducted in 120-mL batch bioreactors containing a nutrient-rich growth medium (ATCC 1977; Van Ooteghem et al., Biotechnol. Lett., 2004, 26:1223) additioned with glucose, molasses or cheese whey as carbon source. For all the substrates tested, T. neapolitana resulted the best-performing strain under suspended-cell conditions, with a 0.9-1.9 mmol / L medium / h H2 production rate at an 8-10 g/L initial substrate concentration, and a 1.6-2.6 mmol H2 / mmol monosaccharide consumed yield. To compare the 4 strains also under attached-growth conditions, preliminary tests were conducted with glucose-growing T. neapolitana, with the goal to select the best biomass carrier among 4 porous materials utilized in biofiltration applications. The best results were obtained with the carrier characterized by the highest interfacial area, equal to 2.1 m2 g-1. Also under attached-growth conditions, T. neapolitana resulted the best strain for all the 3 substrates tested, with a 1.2-1.8 mmol / L medium / h H2 production rate. Further tests, aimed at simplifying the growth medium composition, led to encouraging results. For example, in the case of molasses a minimum medium composed only by NH4Cl, K2HPO4, NaCl, buffer and cysteine resulted – in comparison with the ATCC 1977 complete medium – in a 73% reduction of medium cost and in a 12% increase of the H2/substrate yield. Finally, the process scale-up to a 19-L reactor is in progress. The preliminary results indicate that, in the scale-up of batch H2 production from glucose by T. neapolitana, the values of the three monitored yields (H2/substrate, organic acids/substrate, biomass/substrate) did not show significant variations, whereas the H2 volumetric production and the initial H2 production rate resulted to be significantly affected by the concentration of the initial inoculum.

Bio-hydrogen production from organic residues is an attractive process that combines energy generation with waste treatment. This work describes hydrogen production from glucose, molasses and cheese whey by 4 hyperthermophilic Thermotoga strains. In experiments with glucose-fed T. neapolitana, the best H2 rate was obtained with the biofilm support characterized by the highest specific surface. All the 4 Thermotoga strains examined (T. neapolitana, T. maritima, T. naphtophila, T. petrophila) could produce H2 from glucose, molasses and cheese whey under both suspended- and attached-cell conditions. With all the three substrates, T. neapolitana resulted the best H2 producer. The best H2 production rate was higher on molasses (1.7 mmol H2 / L medium / h) than on cheese whey (0.94 mmol H2 / L medium / h), whereas the highest H2/substrate yields were similar between the two substrates.

Questo lavoro descrive la produzione di idrogeno per digestione anaerobica di siero di latte, glucosio e melasso utilizzando 4 ceppi Thermotoga termofili. Tutte i 4 ceppi di Thermotoga testati (T. neapolitana, T. maritima, T. naphtophila, T. petrophila) sono stati in grado di produrre H2 dal siero di latte, glucosio e melasso, sia in prove con biomassa sospese che adesa. Con tutti i tre substrati, le migliori prestazioni sono state ottenute con T. neapolitana. Sono state condotte alcuni test per selezionare il supporto ottimale per le condizioni con biomassa adesa. Sono stati testati 4 tipi di supporti, 3 in vetro sinterizzato e uno in materiale ceramico; il supporto scelto è stato Biomax.

Chlorinated Aliphatic Hydrocarbons (CAHs) are common contaminants of groundwaters and industrial wastewaters. Several lab- and field-scale studies showed that aerobic cometabolism with aliphatic and aromatic hydrocarbons as growth substrates can lead to the rapid and complete dechlorination of a wide range of CAHs, including high-chlorinated compounds such as tetrachloroethylene. In this study, a process of chloroform (CF) aerobic cometabolic biodegradation by a butane-growing consortium was investigated for 354 days in a 2-m continuous-flow sand-filled reactor. The study was aimed at a) investigating the oxygen/substrate pulsed injection technique as a tool to control the clogging of the porous medium, to attain a long bioreactive zone and to reduce substrate inhibition on CF cometabolism; b) identifying guidelines for optimizing the oxygen/substrate supply schedule; c) developing a reliable model of CF cometabolism in porous media. While the continuous supply of butane rapidly led to the clogging of the porous medium due to excessive biomass growth near the reactor inlet, the testing of six types of oxygen/substrate pulsed feeding led to the identification of a feeding schedule capable to prevent aquifer clogging and to lead to the development of a long bioreactive zone and to satisfactory CF degradation performances (CF removal = 82%; CF degradation rate = 0.035 mg Lreactor d-1; ratio of CF degraded to butane consumed (CF/B) = 0.27 mg mg-1). The tested model of aerobic cometabolism in porous media led to a suitable interpretation of the experimental data (average percent error = 19%) as long as the CF/B ratio was ≤ 0.27 mg mg-1. A long-term simulation of the best-performing schedule of pulsed oxygen/substrate supply indicated the attainment of a steady state condition characterized by lower bioremediation performances than those obtained experimentally, evidencing the need for a further, model-based optimization of the pulsed injection technique.

Trattasi del primo report sulla annotazione del genoma del batterio Pseudomonas pseudoalcaligenes KF707. I dati sono stati sottoposti a pubblicazione on-line e saranno presto messi a disposizione di tutti i ricercatori interessati alla mappa genenica di questo microrganismo

This work focuses on chloroform (CF) cometabolism by a butane-grown aerobic pure culture (Rhodococcus aetherovorans BCP1) in continuous-flow biofilm reactors. The goals were to study CF biodegradation by BCP1 and to evaluate the applicability of the pulsed injection of growth substrate and oxygen in biofilm reactors. The attached-cell tests were initially conducted in a 0.165 L bioreactor and then scaled-up to a 1.772 L bioreactor. Glass cylinders were utilized as biofilm carriers. The continuous supply of growth substrate (butane), which led to the attainment of the highest CF degradation rate (8.2 mg CF / (day m2 biofilm surface)), was compared with 4 schedules of butane and oxygen pulsed feeding. The pulsed injection technique allowed the attainment of a ratio of CF mass degraded per unit mass of butane consumed equal to 0.17 mg CF / mg butane, a value 4 times higher than that obtained with the continuous substrate supply.

Sustainable hydrogen production is a topic of great interest since hydrogen is considered a clean, high-energy content and a low emission fuel. According to this, biological hydrogen production, especially from organic residues, is an attractive process, combining clean energy generation with waste material recycling[1]. Since hydrogen fermentative production at high temperature benefits of general and thermodynamics advantages, hyperthermophilic Thermotoga species are considered ideal organisms for H2 production[2]. The aim of this study is to investigate the feasibility of a cost-effective process of biological H2 production from food industry wastes under thermophilic conditions. The H2-producing performances of 4 Thermotoga strains (Th. neapolitana, Th. petrophila, Th. naphtophila, Th. maritima) were compared at 77 °C by means of tests conducted in 120-mL batch bioreactors containing 40 mL of a nutrient-rich growth medium (ATCC 1977)[3] supplemented with glucose, molasses or whey as main carbon sources. For all the substrates tested, Th. neapolitana resulted the best-performing strain, with a H2 specific production rate of 0.50-0.68 mmol gdry weight-1 h-1 at a 10 g L-1 initial substrate concentration. The H2-producing capacities of the 4 strains were also evaluated under biofilm-growth conditions by using a porous ceramic support utilized in the field of biofiltration as biomass carrier. Also under attached-growth conditions, Th. neapolitana resulted the best strain for all the 3 substrates tested, with a H2 specific production rate of 0.09-0.18 mmol gdry weight-1 h-1. Further tests were aimed at optimizing the growth medium composition. As a result, a significantly more simple – and thus more economical – medium was obtained that allowed the development of a more cost-effective process, even though the resulting H2 production rates were lower than those obtained by growing Thermotoga on the rich medium ATCC 1977. Finally, the preliminary results of a kinetic study of H2 production by attached cells of Th. neapolitana with glucose, molasses and whey indicated a substrate inhibition effect above 20 g L-1 for glucose and whey, but not for molasses.

This work represents the first step of a wider study aimed at evaluating the feasibility of a cost-effective process of biological H2 production from food industry wastes under thermophilic conditions. The H2-producing performances of 4 Thermotoga strains (Th. neapolitana, Th. petrophila, Th. naphtophila, Th. maritima) were compared at 77 °C by means of tests conducted in 120-mL batch bioreactors containing 40 mL of a nutrient-rich growth medium (ATCC 1977; Van Ooteghem et al., Biotechnol. Lett., 2004, 26:1223) additioned with glucose, molasses or whey as carbon source. For all the substrate tested, Th. neapolitana resulted the best-performing strain, with a 0.50-0.68 mmol gdry weight-1 h-1 H2 specific production rate at a 10 g L-1 initial substrate concentration. To compare the H2-producing capacities of the 4 strains also under attached-growth conditions, preliminary tests were conducted with Th. neapolitana, with the goal to select the best biomass carrier among 4 porous materials utilized in the field of biofiltration. The best results were obtained with a porous ceramic product characterized by a 9 m2 g-1 interfacial area. Also under attached-growth conditions, Th. neapolitana resulted the best strain for all the 3 substrates tested, with a 0.09-0.18 mmol gdry weight-1 h-1 H2 specific production rate. Further tests aimed at optimizing the growth medium composition allowed the attainment of H2 production rates slightly lower than the above-reported values, with a significantly more simple – and thus more economical – medium composition. The preliminary results of a kinetic study of H2 production by attached cells of Th. neapolitana with glucose, molasses and whey indicate a substrate inhibition effect above 20 g L-1 for glucose and whey, but not for molasses. Finally, the process scale-up to an innovative 19-L attached-cell agitated reactor is in progress.

This work focuses on chloroform (CF) cometabolism by a butane-grown aerobic pure culture (Rhodococcus aetherovorans BCP1) in continuous-flow attached-growth bioreactors. The goals of this study were: i) to obtain preliminary information on the feasibility of CF biodegradation by BCP1 in biofilm reactors; and ii) to evaluate the applicability of the pulsed injection of growth substrate and oxygen to attached-growth bioreactors. Biofilm of BCP1 was formed on glass cylinders. The continuous supply of growth substrate (butane), which led to the attainment of the highest CF degradation rate, was compared with 4 schedules of butane and oxygen pulsed feeding. The best-performing pulsed injection schedule allowed the attainment of a 60% higher CF conversion (38%) and of a 4-fold increase of the mass of CF degraded per unit mass of butane consumed (0.17), in comparison with the continuous supply of substrate.