M.A. Longo

Ionic liquid-based aqueous biphasic system for lipase extraction

A successful process to extract lipolytic enzymes based on an aqueous biphasic system (ABS), which uses both ionic liquids (ILs) and a high charge-density inorganic salt (K2CO3), is proposed in this work. The activity of a model Thermomyces lanuginosuslipase (TlL) in some of the most common hydrophilic ILs, based on the 1-alkyl-3-methylimidazolium cation, combined with chloride, alkylsulfate, alkylsulfonate and acetate, was investigated. Several operating conditions influencing lipase activity and ABS formation were investigated. Parameters such as temperature, pH, deactivation kinetics and water content were evaluated in order to propose a viable extraction process. A deeper analysis in terms of enzyme deactivation kinetics was carried out, and the data were modelled through a series-type deactivation equation. ATR-FTIR studies aimed at identifying the TlL structure in selected ILs have also provided an insight into the enzyme deactivation behaviour.

Decolorization of dye Reactive Black 5 by newly isolated thermophilic microorganisms from geothermal sites in Galicia (Spain)

In this study, thermophilic microbial strains from thermal spots in northwestern Spain displaying excellent decolorization capability were isolated. The research work tackled: (i) the ability of consortia to degrade a model di-azo dye Reactive Black at different pHs in flask cultures, obtaining that just neutral pHs licensed degradation levels near to 70%, (ii) the isolation of tree of the bacteria, which rendered possible reaching high levels of decolorization (80%) after just 24 h in aerobic conditions, and which were identified through 16S rRNA sequencing to possess high homology (99%) with Anoxybacillus pushchinoensis, Anoxybacillus kamchatkensis and Anoxybacillus flavithermus, and (iii) the cultivation of the isolates in a bench-scale bioreactor, which led to a decolorization rate two-fold higher than that obtained in flask cultures. Therefore, this work makes up the first time that a decolorization process of an azo dye by thermophilic microorganisms in aerobic conditions is investigated.

Strategies for improving extracellular lipolytic enzyme production by Thermus thermophilus HB27

In Thermus thermophilus HB27 cultures the localisation of lipolytic activity is extracellular, intracellular and membrane bound, with low percentage for the former. Therefore, the extracellular secretion must be increased in order to simplify the downstream process and to reduce the economic cost. This study focuses on the design of an innovative operational strategy to increase extracellular lipolytic enzyme production by T. thermophilus HB27 at bioreactor scale. In order to favour its secretion, the effect of several operational variables was evaluated. Among them, the presence of oils in the culture medium leads to improvements in growth and lipolytic enzyme activity. Sunflower oil is the most efficient inducer showing better results when added after 10h of growth. On the other hand, although surfactants lead to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. Thus, by addition of surfactants at the stationary phase, a release of intracellular and membrane enzyme which increases the extracellular enzyme proportion is detected. Based on these results, strategies with successive addition of oil and surfactant in several culture phases in shake flask are developed and verified in a laboratory scale stirred tank bioreactor.

Impact of ionic liquids on extreme microbial biotypes from soil

This work aims at identifying, amongst extreme soil biotypes at locations of high salinity and high hydrocarbon load, microbial strains able to survive short or long-term exposure to the presence of selected ionic liquids. We have evaluated the impact of ionic liquids on the diversity of the soil microbiota to identify which microbial strains have higher survival rates towards ionic liquids, and consequently those which might possibly play a major role in their biotic fate. To the best of our knowledge, this is the first study of this kind. Soils, from a region in Portugal (Aveiro) were sampled and the bacterial and fungal strains able to survive after exposure to high concentrations of selected ionic liquids were isolated and further characterised. We have mainly focused on two types of cations: imidazolium – the most commonly used; and cholinium – generally perceived as benign. The surviving microbial strains were isolated and taxonomically identified, and the ionic liquid degradation was analysed during their cultivation. The continuing exposure of the microbial strains to petroleum hydrocarbons is likely to be the basis for their acquired resistance to some imidazolium salts; also, the higher capacity of fungi – compared to bacteria – to grow, even during their exposure to these liquid salts, became evident in this study.

Technoeconomic assessment of phenanthrene degradation by Pseudomonas stutzeri CECT 930 in a batch bioreactor.

Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent pollutants that accumulate in natural environment mainly as a result of anthropogenic activities. Therefore, the improvement of the available bank of microbial resources and information is crucial to the proper management of PAHs-polluted sites and effluents. In this work, Pseudomonas stutzeri CECT 930 was selected for aerobically degrading an aqueous effluent containing phenanthrene (PHE). Maximum PHE degradation of 90% was obtained both at flask and stirred tank bioreactor scale. All the experimental data were fitted to logistic and Luedeking and Piret models, and licensed to quantitatively ascertain a stronger dependence on the biomass of the metabolites triggering the bioremediation process. In addition, PHE degradation via protocatechuate pathway was elucidated through GC-MS data. Finally, based on the promising results of biodegradation, a preliminary economic evaluation of this process at industrial scale was approached by means of simulation data obtained with SuperPro Designer.

Production of Thermostable Lipolytic Activity by Thermus Species

A quantitative screening for intra‐ and extracellular lipolytic activity was performed in submerged cultures of four Thermus strains using two different media (named T or D medium). Major differences in the extracellular lipolytic activity were observed in T medium, the highest values being for Thermus thermophilus HB27 and Thermus aquaticus YT1 strains (18 and 33 U/L, respectively). Two enzymes with lipase/esterase activity were identified in the four Thermus strains by zymogram analysis, with molecular weights of 34 and 62 kDa. No kinetic typification of the enzymes as primary metabolites was possible for any of the Thermus strains, because of the lack of a good fitting of the experimental lipolytic activity production rates to the Luedecking and Piret model. However, a linear relationship was found between the absolute values of biomass and total lipase/esterase activity (sum of intracellular and extracellular). For T. thermophilus HB27, an increase in the aeration rate caused the increase in the production of biomass and, particularly, intracellular lipolytic activity but the extracellular lipolytic activity was not affected except for the series with the strongest oxygen limitation. Transmission electronic microscopy revealed that T. thermophilus HB27 formed rotund bodies surrounded by a common membrane in cultures in the early stationary phase. The results suggest the occurrence of a specific mechanism of lipase/esterase secretion that might be due to the different composition and permeability of the cell membranes and those surrounding the rotund bodies.

Hydrogen peroxide biosensor with a supramolecular layer-by-layer design.

A new sensor design is reported for the construction of an amperometric enzyme biosensor toward H (2)O(2). It was based in the supramolecular immobilization of alternating layers of horseradish peroxidase (either modified with 1-adamantane or beta-cyclodextrin-branched carboxymethylcellulose residues) on Au electrodes coated with polythiolated beta-cyclodextrin polymer. The analytical response of the electrodes, using 1 mM hydroquinone as an electrochemical mediator, increases when the number of enzyme layers increases. The biosensor having three enzyme layers showed a sensitivity of 720 microA/M cm (2) and a detection limit of 2 microM and retained 96% of its initial activity after 30 days of storage. The host-guest supramolecular nature of the immobilization method was confirmed by cyclic voltammetry.

Comparison between the protease production ability of ligninolytic fungi cultivated in solid state media

Solid state cultures of two white-rot fungi, Phanerochaete chrysosporium and Phlebia radiata, have been carried out, using an inert support (nylon sponge) and a support-substrate (corncob). The suitable medium and culture conditions have been chosen to favour the secretion of ligninolytic enzymes. The production of manganese peroxidase, lignin peroxidase, laccase and proteases has been monitored during the cultures, in an attempt to investigate the possible effect of the latter on the integrity of ligninolytic enzymes. The higher the protease concentration in the culture medium, the more irregular the profiles of ligninolytic enzyme activity. P. chrysosporium secretes proteolytic enzymes mainly during primary metabolism, while P. radiata produced these at the onset of secondary metabolism. Furthermore, different types of proteases produced were identified, P. chrysosporium secreted mainly thiol and acidic proteases, while P. radiata cultures contained thiol-, serin- and metalloproteases.

Performance of a solid-state immersion bioreactor for ligninolytic enzyme production: evaluation of different operational variables

The production of ligninolytic enzymes by the white-rot fungus Phanerochaete chrysosporium BKM-F-1767 in a solid-state immersion bioreactor, employing cubes of nylon sponge as a support, was studied. Cultivation was carried out in both batch and continuous mode, and the effect of some operational variables (aeration level, pH) was investigated. Batch operation at an aeration level of 0.5 vvm led to maximum manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 574 and 116 U l−1, respectively. The results were compared with those obtained at a higher aeration level (1 vvm), reported in a previous work, and it appeared that LiP productivity increased with aeration rate, while MnP was not significantly affected. Continuous operation showed much lower MnP activities (around 60 U l−1) and similar LiP activities (about 132 U l−1). However, in terms of productivity the difference between batch and continuous operation for MnP was less remarkable (239 and 150 U day−1, respectively), whereas LiP productivity was ten-fold higher in continuous operation than in batch mode. This could be attributed to the influence of operation pH on ligninolytic enzyme activities. The study of the kinetic characteristics of the biocatalysts supported this hypothesis.

Assessment of a process to degrade metal working fluids using Pseudomonas stutzeri CECT 930 and indigenous microbial consortia

The development of a novel biological process to treat metal working fluids (MWFs)-containing effluents at bioreactor scale was pursued in this work. The bacteria Pseudomonas stutzeri CECT 930 was investigated for the first time as an alternative agent for MWF degradation. An adequate medium design and mixing and aeration system, as well as an appropriate microorganism proved to be crucial for reaching high levels of degradation by P. stutzeri and by an indigenous consortium (about 70% and 50% of reduction in total petroleum hydrocarbon content in less than 2 wk, respectively). Additionally, as there is no information in literature trying to kinetically characterize an MWF-polluted effluent degradation process, all the experimental data were fitted to logistic and Luedeking and Piret models, that allowed to elucidate the growth-associated character of the biodegradation process.