Ilaria Finore

Fermentation technologies for the optimization of marine microbial exopolysaccharide production.

In the last decades, research has focused on the capabilities of microbes to secrete exopolysaccharides (EPS), because these polymers differ from the commercial ones derived essentially from plants or algae in their numerous valuable qualities. These biopolymers have emerged as new polymeric materials with novel and unique physical characteristics that have found extensive applications. In marine microorganisms the produced EPS provide an instrument to survive in adverse conditions: They are found to envelope the cells by allowing the entrapment of nutrients or the adhesion to solid substrates. Even if the processes of synthesis and release of exopolysaccharides request high-energy investments for the bacterium, these biopolymers permit resistance under extreme environmental conditions. Marine bacteria like Bacillus, Halomonas, Planococcus, Enterobacter, Alteromonas, Pseudoalteromonas, Vibrio, Rhodococcus, Zoogloea but also Archaea as Haloferax and Thermococcus are here described as EPS producers underlining biopolymer hyperproduction, related fermentation strategies including the effects of the chemical composition of the media, the physical parameters of the growth conditions and the genetic and predicted experimental design tools.

The hemicellulose extract from Cynara cardunculus: a source of value-added biomolecules produced by xylanolytic thermozymes

The Cynara cardunculus hemicellulose fraction was recovered from its stem and leaf biomass and converted into valuable molecules by exploiting the extracellular xylanase and β-xylosidase activities produced by the thermophilic bacterium Geobacillus thermantarcticus. Several degradation procedures for the arabinoglucuronoxylan extract were proposed by using efficient and different enzymatic preparations, containing both or only one xylanolytic activity of G. thermantarcticus. In particular, when the xylanase and β-xylosidase activities were used separately in hydrolysis reactions, xyloglucurono-oligosaccharides or xylose were obtained with a yield of 32% and 62.6% respectively, with reference to the hemicellulosic extract. Furthermore, the synergic action of β-xylosidase/xylanase activities was exploited in transglycosylation processes for the production of xylo-conjugated compounds; xylosides of primary alcohols with increasing carbon chains and aromatic alcohols were produced starting from the C. cardunculus hemicellulose, which was selected as a cheap donor. When 2-phenoxyethanol was selected as an acceptor, 2-phenoxyethyl β-xyloside, xylobioside and xylotrioside were prepared with a yield of 38.5% with respect to the hemicellulosic extract and spectroscopically characterized.

Production of succinic acid from Basfia succiniciproducens up to the pilot scale from Arundo donax hydrolysate.

In the present work the recently isolated strain Basfia succiniciproducens BPP7 was evaluated for the production of succinic acid up to the pilot fermentation scale in separate hydrolysis and fermentation experiments on Arundo donax, a non-food dedicated energy crop. An average concentration of about 17g/L of succinic acid and a yield on consumed sugars of 0.75mol/mol were obtained demonstrating strain potential for further process improvement. Small scale experiments indicated that the concentration of acetic acid in the medium is crucial to improve productivity; on the other hand, interestingly, short-term (24h) adaptation to higher acetic acid concentrations, and strain recovery, were also observed.

Microbial Diversity in Extreme Marine Habitats and Their Biomolecules

Extreme marine environments have been the subject of many studies and scientific publications. For many years, these environmental niches, which are characterized by high or low temperatures, high-pressure, low pH, high salt concentrations and also two or more extreme parameters in combination, have been thought to be incompatible to any life forms. Thanks to new technologies such as metagenomics, it is now possible to detect life in most extreme environments. Starting from the discovery of deep sea hydrothermal vents up to the study of marine biodiversity, new microorganisms have been identified, and their potential uses in several applied fields have been outlined. Thermophile, halophile, alkalophile, psychrophile, piezophile and polyextremophile microorganisms have been isolated from these marine environments; they proliferate thanks to adaptation strategies involving diverse cellular metabolic mechanisms. Therefore, a vast number of new biomolecules such as enzymes, polymers and osmolytes from the inhabitant microbial community of the sea have been studied, and there is a growing interest in the potential returns of several industrial production processes concerning the pharmaceutical, medical, environmental and food fields.

Nesterenkonia aurantiaca, sp. nov., an alkaliphilic actinobacterium isolated from Cape King (Antarctica).

A Gram-stain-positive, non-endospore-forming, haloalkaliphilic actinobacterium, strain CK5T, was isolated from a soil sample, collected at Cape King (Antarctica), and its taxonomic position was investigated by using a polyphasic approach. Cells were cocci with orange pigmentation, non-motile and grew optimally at 25 °C and pH 9.0-9.5 in the presence of 2 % (w/v) NaCl. Cellular membrane contained MK-7 (72 %) and MK-8 (28 %), and anteiso-C15 : 0 (64.8 %), iso-C16 : 0 (13.3 %), n-C17 : 0 (9.9 %), n-C16 : 0 (4.0 %), n-C14 : 0 (3.7 %) as major cellular fatty acids. The DNA G+C content was 64.8 mol%. Strain CK5T, based on the 16S rRNA gene sequence similarity, was most closely related to Nesterenkonia jeotgali JG-241T (99.5 %), Nesterenkonia sandarakina YIM 70009T (99.4 %), Nesterenkonia lutea YIM 70081T (99.4 %), Nesterenkonia halotolerans YIM 70084T (99.3 %), Nesterenkonia xinjiangensis YIM 70097T (97.2 %), Nesterenkonia flava CAAS 251T (97.1 %) and Nesterekonia aethiopica CCUG 48939T (97.1 %). Strain CK5T revealed 31 % DNA-DNA relatedness with respect to N. sandarakina DSM 15664T, 29 % with respect to N. jeotgali DSM 19081T, 10 % with respect to N. lutea DSM 15666T and 1 % with respect to N. halotolerans, DSM 15474T, N. xinjiangensis DSM 15475T, N. aethiopica DSM 17733T and N. flava DSM 19422T. On the basis of 16S rRNA gene sequences, DNA-DNA hybridization and chemotaxonomic characteristics, strain CK5T represents a novel species of the genus Nesterenkonia, for which the name Nesterenkonia aurantiaca sp. nov. is proposed. The type strain is CK5T ( = DSM 27373T = JCM 19723T).

Degradative actions of microbial xylanolytic activities on hemicelluloses from rhizome of Arundo donax.

Polysaccharidases from extremophiles are remarkable for specific action, resistance to different reaction conditions and other biotechnologically interesting features. In this article the action of crude extracts of thermophilic microorganisms (Thermotoga neapolitana, Geobacillus thermantarcticus and Thermoanaerobacterium thermostercoris) is studied using as substrate hemicellulose from one of the most interesting biomass crops, the giant reed (Arundo donax L.). This biomass can be cultivated without competition and a huge amount of rhizomes remains in the soil at the end of cropping cycle (10–15 years) representing a further source of useful molecules. Optimization of the procedure for preparation of the hemicellulose fraction from rhizomes of Arundo donax, is studied. Polysaccharidases from crude extracts of thermophilic microorganisms revealed to be suitable for total degradative action and/or production of small useful oligosaccharides from hemicelluloses from A. donax. Xylobiose and interesting tetra- and pentasaccharide are obtained by enzymatic action in different conditions. Convenient amount of raw material was processed per mg of crude enzymes. Raw hemicelluloses and pretreated material show antioxidant activity unlike isolated tetra- and pentasaccharide. The body of results suggest that rhizomes represent a useful raw material for the production of valuable industrial products, thus allowing to increase the economic efficiency of A. donax cultivation.

Production and Biotechnological Potential of Extracellular Polymeric Substances from Sponge-Associated Antarctic Bacteria

ABSTRACT Four sponge-associated Antarctic bacteria (i.e., Winogradskyella sp. strains CAL384 and CAL396, Colwellia sp. strain GW185, and Shewanella sp. strain CAL606) were selected for the highly mucous appearance of their colonies on agar plates. The production of extracellular polymeric substances (EPSs) was enhanced by a step-by-step approach, varying the carbon source, substrate and NaCl concentrations, temperature, and pH. The EPSs produced under optimal conditions were chemically characterized, resulting in a moderate carbohydrate content (range, 15 to 28%) and the presence of proteins (range, 3 to 24%) and uronic acids (range, 3.2 to 11.9%). Chemical hydrolysis of the carbohydrate portion revealed galactose, glucose, galactosamine, and mannose as the principal constituents. The potential biotechnological applications of the EPSs were also investigated. The high protein content in the EPSs from Winogradskyella sp. CAL384 was probably responsible for the excellent emulsifying activity toward tested hydrocarbons, with a stable emulsification index (E24) higher than those recorded for synthetic surfactants. All the EPSs tested in this work improved the freeze-thaw survival ratio of the isolates, suggesting that they may be exploited as cryoprotection agents. The addition of a sugar in the culture medium, by stimulating EPS production, also allowed isolates to grow in the presence of higher concentrations of mercury and cadmium. This finding was probably dependent on the presence of uronic acids and sulfate groups, which can act as ligands for cations, in the extracted EPSs. IMPORTANCE To date, biological matrices have never been employed for the investigation of EPS production by Antarctic psychrotolerant marine bacteria. The biotechnological potential of extracellular polymeric substances produced by Antarctic bacteria is very broad and comprises many advantages, due to their biodegradability, high selectivity, and specific action compared to synthetic molecules. Here, several interesting EPS properties have been highlighted, such as emulsifying activity, cryoprotection, biofilm formation, and heavy metal chelation, suggesting their potential applications in cosmetic, environmental, and food biotechnological fields as valid alternatives to the commercial polymers currently used.

Use of Agro Waste Biomass for α-Amylase Production by Anoxybacillus amylolyticus: Purification and Properties

Knowledge accumulated from fundamental and applied studies on Anoxybacillus suggests that this genus can serve as a good alternative in many applications related to starch and lignocellulosic biomasses, waste treatment, enzyme technology, and bioenergy production. We investigated the purification, biochemical characterization and immobilization of a thermostable α-amylase from the thermophilic Anoxybacillus amylolyticus, strain MR3CT, isolated in Antarctica and its production on vegetable wastes. In particular, the rhizome from Arundo donax, waste biomass of Cynara cardunculus and potato peels were tested either in Submerged Fermentation (SmF) and Solid State Fermentation (SSF) conditions. The amylase from A. amylolyticus, with a molecular weight of about 60 kDa, displayed an optimum enzyme activity at 60°C and pH 5.6. Moreover, by retaining up to 70% of total activity after 48 h at 60°C, it showed high thermostability in the presence of 2 mM calcium ion. The immobilized enzyme maintained the 48% of its initial activity after the sixth reuse. The optimal conditions for its production in SmF were achieved at 60°C for 24 h with 1% of rhizome from Arundo donax, which was about 2126 U/gds. SSF cultures reached maximum α-amylase yield (102 U/gds) when grown on waste biomass of Cynara cardunculus as substrate, with a substrate-water ratio of 1:1 (w/v), and incubation at 60°C for 4 days. In this study, rhizome of A. donax resulted to be a good substrate for amylase production in SmF thus allowing a cheaper alternative to obtain amylolytic enzymes. Indeed by using rhizomes from A. donax as growth substrate it was possible to recovery an amylase activity level higher than that obtained by synthetic complex medium. Amylase production was also investigated under SSF conditions by using the above listed wastes as sole carbon source for A. amylolyticus growth. Under these conditions, C. cardunculus gave a higher enzyme yield per reactor volume.

Biotechnology Implications of Extremophiles as Life Pioneers and Wellspring of Valuable Biomolecules

Studies on extremophiles, microorganisms able to survive in extreme environments, are very helpful for the comprehension of life evolution; in fact they are the unique organisms of the Earth at the origin of life. They lie into the three domains of life (Archaea, Bacteria, and Eukarya) and can be found in environmental niches on Earth such as in hydrothermal vents and springs, in salty lakes, in halite crystals, in polar ice and lakes, in volcanic areas, in deserts, or under anaerobic conditions. The existence of life forms beyond the Earth requires an extension of the classical limits of life: the resistance of extremophilic organisms to harsh conditions in terms of temperature, salinity, pH, pressure, dryness, and desiccation makes these living organisms good putative candidates to assess the habitability of other planets. The ability to survive and proliferate in extreme conditions (pH, temperature, pressure, salt, and nutrients) produces a variety of biotechnologically useful molecules such as lipids, enzymes, polysaccharides, and compatible solutes that are employed in several industrial processes. There are many extremophilic enzymes and also endogenous compounds that are used with success for food industry, for preparation of the detergents, for pharmacological applications, and also for genetic studies. In particular enzymes that derive from thermophiles, and for this reason called thermozymes, represent an excellent sources of new catalysts of interest in industrial sectors.

Technical Developments for Vegetable Waste Biomass Degradation by Thermophiles

Environmental concerns such as greenhouse gas emission and fossil fuels depletion, have driven increasing technological and economical interest in the study of biorefining processes in order to convert organic waste materials (biomass) into bioethanol, biodiesel, building blocks (chemicals) and biomaterials. Vegetable waste biomass is produced continuously at global level by agro-industries, agriculture and forestry and according to its origin, it can be distinguished in three main groups i.e.: food wastes, crop residues and forestry/wood residues. Plant biomass and vegetable industrial wastes such as lignocellulosic biomass is object of great interest since it can be hydrolysed to have starch, (hemi)cellulose and lignin that in turn will be converted in value added chemicals and/or biofuels. Pre-treatment steps using physico-chemical or enzymatic processes, make the conversion of lignocellulosic biomass into biofuels more expensive than the extraction of fossil fuels. This chapter underlines the capability of thermophiles and of their enzymes to bypass the problems and limits linked with the lignocellulosic biomass use. Studies concerning the exploitation of agro-waste as growth medium for the production of biotechnologically useful extremophilic microorganisms and their relative enzymes, the pre-treatment and digestion of lignocellulosic fractions in order to obtain mono- and oligosaccharides, the use of thermophilic enzymes in comparison to that of commercial, for a convenient set up of a total degrading process, the chemical procedures for the characterization of new compounds obtained from lignocellulosic materials, are also discussed. Moreover, the opportunity to employ thermophiles in the conversion of lignocellulosic materials into ethanol using only one step process, are also provided.