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1 DICAM, University of Bologna, via Terracini 28, I-40131, Bologna, Italy 2 Eridania Sadam S.p.A, Via degli Agresti 4/6, I-40123, Bologna, Italy 3 Sebigas S.p.A., Via Santa Rita, 21057, Olgiate Olona (VA), Italy 4 Sadam Engineering s.r.l., Via degli Agresti 4/6, I-40123, Bologna, Italy Stefano Rebecchi1, Lorenzo Bertin1, Veronica Vallini2, Giacomo Bucchi3, Fabrizio Bartocci4, Fabio Fava1 Rimini, 7 Novembre 2013

Il lavoro di Dottorato si è incentrato con successo sullo studio della possibilità di applicare il modello ADM1 per la descrizione e verifica di impianti industriali di digestione anaerobica. Dai dati sperimentali il modello e l'implementazione in software di analisi numerica si sono rivelati strumenti efficaci. Il software sviluppato è stato utilizzato come strumento di progettazione di impianti alimentati con biomasse innovative, analizzate con metodiche biochimiche (BMP) in scala di laboratorio. Lo studio è stato corredato con lo studio di fattibilità di un impianto reale con verifica di ottimo economico., This work of thesis succesfully studied the feasibility of ADM1 application to Anaerobic Digestion industrial biogas plant description. The model and its implementation in numerical analysis software is an efficient tool. The software was applied to new substrates tested in bench scale tests (BMP tests) to design innovative AD bioreactors on industrial scale. This study also features the feasibility study of an existing industrial biogas plant, completed with the economic study (business plan).

The aim of this study was to develop a packed bed reactor (PBR) process for the on-site aerobic cometabolic bioremediation of a groundwater contaminated by trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA). With regard to the growth substrate selection, the comparison of the CAH degradation performances obtained with 5 candidate substrates led to the selection of butane and to the development from the site’s indigenous biomass of a suspended-cell consortium capable to degrade TCE (TCE first-order constant = 96 L gprotein-1 d-1 at 30 °C and 4.3 L gprotein-1 d-1 at 15 °C) with a 90% mineralization of the organic Cl. Based on PCR-DGGE analysis of the 16S rRNA genes followed by band excision and sequencing, the microbial consortium enriched was mainly composed of Bacteroidetes and Alpha- and Beta-Proteobacteria that were distantly related to known CAH-cometabolizing bacteria. The low TeCA degradation rate by the developed consortium suggested the introduction of a chemical pre-treatment based on the TeCA to TCE conversion via -elimination, a very fast reaction at alkaline pH. The choice of the best-performing carrier was made by means of a 2-level procedure: the 1st level consisted of batch tests, operated both at 30 and 15 °C, whereas the 2nd level consisted of continuous-flow tests, operated at 30 °C. A porous ceramic carrier named Biomax resulted the best-performing one. On the basis of the kinetic parameters relative to TCE aerobic cometabolic biodegradation by the selected consortium, a 31-L packed-bed reactor, consisting of 14 columns connected in series, packed with Biomax and immerged in a temperature-controlled bath, was designed and set-up. The preliminary data, obtained by operating the plant with a 4-day residence time, indicate the attainment of a 0.1 mg L-1 d-1 TCE biodegradation rate.

A procedure for the design of a packed bed reactor (PBR) process for the on-site aerobic cometabolic bioremediation of a groundwater contaminated by trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA) was developed. The comparison of the CAH degradation performances obtained with five substrates led to the selection of butane and to the development from the site’s indigenous biomass of a consortium capable to degrade TCE, with a 90% mineralization of the organic Cl. A porous ceramic carrier (Biomax) resulted the best option for the PBR process. On the basis of the kinetic analysis, a 31-L PBR, consisting of 14 columns connected in series, was designed.

Chlorinated aliphatic hydrocarbons (CAHs) are widespread groundwater contaminants. Aerobic cometabolism, that requires the supply of a suitable growth substrate, represents an interesting option for the remediation of CAH-contaminated aquifers, thanks to its capability to lead to the complete mineralization of a very wide range of CAHs. The aim of this study was to develop and validate a procedure relative to the lab-scale tests required to obtain the essential information for the design of a process of CAH aerobic cometabolism in a packed bed reactor (PBR). To validate the procedure, the latter was applied to the development of a PBR process for the on-site aerobic cometabolism of an aquifer contaminated by trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA). The specific goals of this study, corresponding to the main steps of the above-mentioned procedure, were: (i) to select the best growth substrate for the aerobic cometabolic process, and to develop and characterize an effective CAH-degrading microbial consortium, obtained from the site’s indigenous biomass by exposition to the selected substrate; (ii) to select the best carrier for the PBR process, and to evaluate the effect of bacterial adhesion on the developed suspended-cell consortium; and (iii) to identify and test suitable chemical-physical remediation alternatives in the case of presence of CAHs poorly biodegradable through AC. With regard to the growth substrate selection, the comparison of the CAH degradation performances obtained with 5 candidate substrates (methane, propane, butane, pentane and phenol) led to the selection of butane and to the development from the site’s indigenous biomass of a suspended-cell consortium capable to degrade TCE (TCE first-order constant (k1,TCE) = 96 L gprotein-1 d-1 at 30 °C and 4.3 L gprotein-1 d-1 at 15 °C) with a 90% mineralization of the organic Cl. Based on PCR-DGGE analysis of the 16S rRNA genes followed by band excision and sequencing, the microbial consortium enriched was mainly composed of Bacteroidetes and Alpha- and Beta-Proteobacteria that were distantly related to known CAH-cometabolizing bacteria. With regard to the selection of the best-performing biofilm carrier, a preliminary screening based on the previous experience of the research group led to the pre-selection of four candidate biofilm carriers (porous materials specifically designed for biofilm processes): Biomax, Biomech, Biopearl and Cerambios. The choice of the best-performing carrier was made by means of a 2-level procedure. The 1st level consisted of batch tests, operated both at 30 and 15 °C, whereas the 2nd level consisted of continuous-flow tests, operated at 30 °C. The 30 °C continuous-flow tests were conducted in four 1 L packed columns, connected to a feeding system designed so as to attain a pulsed feed of both oxygen and the selected growth substrate (butane). The four columns were operated in continuous mode for about 100 days. The results of the attached-cell tests were compared on the basis of the TCE normalized degradation rate ( and ) and of the attached cell concentration attained, at the two temperatures, at the end of the biofilm development process. On the basis of both the batch and the continuous-flow tests, Biomax resulted the best-performing biofilm carrier. Biomass immobilization on the carrier changed remarkably the structure of the microbial consortium. The effect on k1,TCE of biomass attachment depended on temperature: at 15 °C the attached consortium performed slightly better than the suspended one, whereas at 30 °C an opposite trend was noticed. On the basis of a 1st-order simulation, a 99.9% TCE conversion can be attained, at the site’s temperature, with a 9-hour HRT. Lastly, the low TeCA degradation rate by the developed consortium suggested the introduction of a chemical pre-treatment based on the TeCA to TCE conversion via -elimination, a very fast reaction at alkaline pH. On the basis of the overall results, the procedure for the development of a PBR AC process appears to be correctly designed and generally applicable to CAH contaminated sites.

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.

This work focuses on the enrichment of a trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA) cometabolizing aerobic microbial consortium from a contaminated site in Northern Italy, for the development of an on-site bioremediation treatment. The goals were: (i) to select the best growth substrate and the best-performing consortium in terms of contaminant degradation capability; (ii) to perform a preliminary suspended-cell kinetic test; (iii) to perform a preliminary evaluation of the feasibility of the co-metabolic treatment of the tested groundwater in a packed-bed reactor operating at the site’s temperature (15°C). The best degradation results were obtained with a butane-growing consortium. At 15° C, the selected culture resulted in short lag-times for the onset of butane utilization (< 1 day) and TCE and TeCA degradation (< 1 - 8 days). Once immobilized on 4 different porous carriers, the selected consortium was able to grow and form a stable biofilm. The TCE and TeCA degradation rates resulted similar for the 4 carriers tested. Further experiments are in progress to estimate the kinetic parameters of the co-metabolic processes and to understand which carrier leads to the best performances in a continuous-flow packed-bed reactor process.

Chlorinated aliphatic hydrocarbons (CAHs) are toxic and environmental hazardous solvents increasingly detected in soil and groundwater for which new and most effective biormemdiation approaches are needed. In this context, the European project MINOTAURUS is aiming to develop groundwater bioremediation robust and reliable strategies based on the use of immobilized biocatalysts. This work was focused on the enrichment of aerobic trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA) co-metabolizing microbial cultures from a contaminated site in Rho (MI, Italy), their preliminary characterization both as freely suspended and immobilized biomass on different carrier materials (Biomax®, Biopearl®, Biomech®, Cerambios®) and the selection of the best performing culture to be employed in a continuous Packed Bed Reactor (PBR) process. The best performing culture was obtained through serial enrichment on filter sterilized site-water or a similar synthetic water in the presence of butane (2 mg/L) as carbon and energy source and TCE (10 mg/L) plus TeCA (3 mg/L). The structure and composition of the microbial population was carried out through denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes. The DGGE analysis outlined that the structure of the suspended microbial community changed remarkably throughout the enrichment with the exception of the most prominent phylotype. However, according to the Dice similarity coefficient, lower changes occurred during the last sub-culturing step, indicating a gradual stabilization of the community structure. When immobilized on different carriers, the most prominent phylotype previously detected represented a minor fraction of the immobilized community; conversely, several of the minor bands occurring in the suspended culture became dominant within the biofilm. Such a drastic change in the structure of the microbial community was evidenced by the separate clustering of the DGGE profiles from suspended cell cultures and biofilms. Furthermore, the high similarity indexes between the communities immobilized on different carriers (57.3% to 73.7%) suggest that the evolved consortium is quite stable and that the material and shape of the carrier do not affect remarkably its composition. Complementary information obtained from degradation kinetic tests showed that the selected culture immobilized on Biomax® retained the highest degrading activity. Identification of the community members via band sequencing and phylogenetic analysis is in progress and will allow to obtain key information on the potential TCE and TeCA co-metabolizing species both in the suspended and in the immobilized communities.

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.

The aim of this work was to develop a high-performing microbial consortium capable to degrade trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA) via aerobic co-metabolism in packed bed reactors (PBRs). To this goal, five groundwaters were sampled from different monitoring wells in an aquifer contaminated by TCE and TeCA, and 25 119-mL batch tests were set up to test five different growth substrates: methane, propane, butane, pentane and phenol. Substrate and oxygen were periodically re-added. TCE and TeCA were spiked at increasing concentrations in order to select microorganisms able to rapidly degrade TCE and TeCA. Preliminary DGGE analysis showed appreciable differences in the microbial populations enriched. Butane was identified as the best-performing growth substrate, and a high-performing butane-growing consortium (B4) was selected for the subsequent continuous-flow tests. The selection was made on the basis of the substrate, TCE and TeCA specific biodegradation rates. Consortium B4 showed a specific rate of 129 L/g protein/day for TCE and 2.6 L/g protein/day for TeCA. The first value is comparable to the rates attainable in active carbons adsorption processes, with the great advantage of the total mineralization of the chlorinated hydrocarbons (Segar et al., Water Environ. Res. 1995;67:764-774). The highest co-metabolized concentrations were 10 mg/L for TCE and 3 mg/L for TeCA. Specific tests aimed at developing and calibrating a kinetic model of substrate consumption and TCE/TeCA degradation are in progress. Attached-cell preliminary tests showed the ability of consortium B4 to form a stable biofilm on porous carriers. The attached biomass was able to utilize butane as carbon source and to biodegrade TCE and TeCA. In order to select the most suitable biofilm carrier for TCE/TeCA aerobic cometabolism by B4 in a PBR, 4 1-L glass columns were filled with 4 types of porous biofilm carriers. Fluid dynamics tests were performed with oxygen, TCE and TeCA in order to estimate the longitudinal dispersivity, the effective porosity and the TCE/TeCA retardation factor of each tested carrier. Four liters of a suspension of consortium B4 were produced in a fermentor in order to colonize the 4 glass columns. Continuous-flow tests of TCE and TeCA cometabolism by biofilms of consortium B4 are in progress, and will be utilized to select the best-performing carrier.

The general purpose of this study was to develop an aerobic cometabolic process in a packed bed reactor to treat groundwater contaminated by trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA). The specific goals were to select the best performing microbial consortium for the cometabolic process and the best support for biofilm formation. Groundwater samples taken from 5 wells of the contaminated site were fed with 5 candidate growth substrates. Butane was selected as the best substrate. The first-rate constants obtained with butane were 130 L / g protein / d for TCE and 2.6 L / g protein / d for TeCA. As a result of a study conducted both in batch and continuous bioreactors, Biomax® resulted the best carrier among 4 candidate porous carriers.

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.

The present work aimed at the recovery and characterization of polyphenolic compounds extracted from red grape pomace (Vitis vinifera L.), a winemaking by-product. Polyphenolic compounds of wet (WP) and dried (DP) red pomace were recovered by enzymatic digestions and ethanol-based extractions. Fungamyl and Celluclast enzymes were found to be the most effective in enhancing polyphenol release from WP. WP samples showed the highest capacity of releasing polyphenols with 2. h control 24°C and 2. h 1% Celluclast resulting as the best treatments. A significantly lower amount of polyphenols was recovered from DP most probably as a consequence of the pomace drying. The best extracts contained high amounts of total polyphenols, flavonoids, tannins and anthocyanins and exerted antioxidant and cholesterol-lowering activities. The results support the possibility of exploiting the extracts coming from grape processing by-products as ingredients for functional and innovative products in the nutraceutical, pharmaceutical or cosmetic fields.