Publications

Microbial desulfurization has been extensively studied as a promising alternative to the widely applied chemical desulfurization process. Sulfur removal from petroleum and its products becomes essential, as the environmental regulations become increasingly stringent. Rhodococcus qingshengii IGTS8 has gained ground as a naturally occurring model biocatalyst, due to its superior specific activity for desulfurization of dibenzothiophene (DBT). Recalcitrant organic sulfur compounds—DBT included—are preferentially removed by selective carbon-sulfur bond cleavage to avoid a reduction in the calorific value of the fuel. The process, however, still has not reached economically sustainable levels, as certain limitations have been identified. One of those bottlenecks is the repression of catalytic activity caused by ubiquitous sulfur sources such as inorganic sulfate, methionine, or cysteine. Herein, we report an optimized culture medium for wild-type stain IGTS8 that completely alleviates the sulfate-mediated repression of biodesulfurization activity without modification of the natural biocatalyst. Medium C not only promotes growth in the presence of several sulfur sources, including DBT, but also enhances biodesulfurization of resting cells grown in the presence of up to 5 mM sulfate. Based on the above, the present work can be considered as a step towards the development of a more viable commercial biodesulfurization process.

The purine utilization pathway has been thoroughly characterized in Aspergillus nidulans. We establish here the subcellular distribution of seven key intracellular enzymes, xanthine dehydrogenase (HxA), urate oxidase (UaZ), 5-hydroxy-isourate hydrolase (UaX), 2-oxo-4-hydroxy-4-carboxy ureido imidazoline decarboxylase (UaW), allantoinase (AlX), allantoicase (AaX), ureidoglycolate lyase (UglA), and the fungal specific a-ketoglutarate Fe(II)-dependent dioxygenase (XanA). HxA, AlX, AaX, UaW and XanA are cytosolic, while UaZ, UaX and UglA are peroxisomal. Peroxisomal localization was confirmed by using appropriate pex mutants. The pathway is largely, but not completely conserved in the Eurotiomycetes, noticeably in some species AaX is substituted by an alternative enzyme of probable bacterial origin. UaZ and the urate–xanthine UapA and UapC transporters, are also localized in specific cells of the conidiophore. We show that metabolic accumulation of uric acid occurring in uaZ null mutations is associated with an increased frequency of appearance of morphologically distinct colony sectors, diminished conidiospore production, UV resistance and an altered response to oxidation stress, which may provide a rationale for the conidiophore-specific localization. The pathway-specific transcription factor UaY is localized in both the cytoplasm and nuclei under non-inducing conditions, but it rapidly accumulates exclusively to the nuclei upon induction by uric acid.

Background: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism. Results: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source. Conclusions: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics.

The ascomycete Paecillomyces variotii was evaluated for the first time as a candidate species for the production of bioethanol from lignocellulose through consolidated bioprocessing (CBP) approaches. The examined strain (ATHUM 8891) revealed all the necessary phenotypic characteristics required for 2nd generation biofuel production. The fungus is able to efficiently ferment glucose and xylose to ethanol, with yields close to the theoretical maximum. Nitrogen supplementation greatly affected ethanol production with nitrate-nitrogen presenting the best results. Notably, ethanol yield on xylose fermentation was higher than that of glucose, while in co-fermentation of glucose–xylose mixtures no distinguished diauxic behavior was observed. Furthermore, the fungus seems to possess the necessary enzyme factory for the degradation of lignocellulosic biomass, as it was able to grow and produce ethanol on common agro-industrial derivatives. Overall, the results of our study indicate that P. variotii is a new and possibly powerful candidate for CBP applications.

Aims: To examine the biocontrol potential of multiactive Greek indigenous Streptomyces isolates carrying antifungal activity against Rhizoctonia solani that causes damping-off symptoms on beans.Methods and Results: A total of 605 Streptomyces isolates originated from 12 diverse Greek habitats were screened for antifungal activity against R. solani DSM843. Almost one-third of the isolates proved to be antagonistic against the fungus. From the above isolates, six were selected due to their higher antifungal activity, identified by analysing their 16S rRNA gene sequence and studied further. The obtained data showed the following: firstly, the isolates ACTA1383 and ACTA1557 exhibited the highest antagonistic activity, and therefore, they were selected for in vivo experiments using bean seeds as target; secondly, in solid and liquid culture experiments under optimum antagonistic conditions, the medium extracts from the isolates OL80, ACTA1523, ACTA1551 and ACTA1522 suppressed the growth of the fungal mycelium, while extracts from ACTA 1383 and ACTA1557 did not show any activity. Conclusions: These results corresponded important indications for the utility of two Greek indigenous Streptomyces isolates (ACTA1557 and ACTA1383) for the protection of the bean crops from R. solani damping-off symptoms, while four of them (isolates OL80, ACTA1523, ACTA1551 and ACTA1522) seem to be promising producers of antifungal metabolites. Significance and Impact of the Study: This is the first study on the biocontrol of R. solani using multiactive Streptomyces isolates originated from ecophysiologically special Greek habitats. Our study provides basic information to further explore managing strategies to control this critical disease.

In a bioprospecting effort towards novel thermostable lipases, we assessed the lipolytic profile of 101 bacterial strains isolated from the volcanic area of Santorini, Aegean Sea, Greece. Screening of lipase activity was performed both in agar plates and liquid cultures using olive oil as carbon source. Significant differences were observed between the two screening methods with no clear correlation between them. While the percentage of lipase producing strains identified in agar plates was only 17%, lipolytic activity in liquid culture supernatants was detected for 74% of them. Nine strains exhibiting elevated extracellular lipase activities were selected for lipase production and biochemical characterization. The majority of lipase producers revealed high phylogenetic similarity with Geobacillus species and related genera, whilst one of them was identified as Aneurinibacillus sp. Lipase biosynthesis strongly depended on the carbon source that supplemented the culture medium. Olive oil induced lipase production in all strains, but maximum enzyme yields for some of the strains were also obtained with Tween-80, mineral oil, and glycerol. Partially purified lipases revealed optimal activity at 70–80 °C and pH 8-9. Extensive thermal stability studies revealed marked thermostability for the majority of the lipases as well as a two-step thermal deactivation pattern.

The biomass degrading enzymatic potential of 101 thermophilic bacterial strains isolated from a volcanic environment (Santorini, Aegean Sea, Greece) was assessed. 80 % of the strains showed xylanolytic activity in Congo Red plates, while only eight could simultaneously hydrolyze cellulose. Fifteen isolates were selected on the basis of their increased enzyme production, the majority of which was identified as Geobacilli through 16S rDNA analysis. In addition, the enzymatic profile was evaluated in liquid cultures using various carbon sources, a procedure that revealed lack of correlation on xylanase levels between the two cultivation modes and the inability of solid CMC cultures to fully unravel the cellulose degrading potential of the isolates. Strain SP24, showing more than 99 % 16S DNA similarity with Geobacillus sp. was further studied for its unique ability to simultaneously exhibit cellulase, xylanase, β-glucosidase and β-xylosidase activities. The first two enzymes were produced mainly extracellularly, while the β-glycosidic activities were primarily detected in the cytosol. Maximum enzyme production by this strain was attained using a combination of wheat bran and xylan in the growth medium. Bioreactor cultures showed that aeration was necessary for both enhanced growth and enzyme production. Aeration had a strong positive effect on cellulase production while it negatively affected expression of β-glucosidase. Xylanase and β-xylosidase production was practically unaffected by aeration levels.

Xanthan gum is a polysaccharide that is widely used as stabilizer and thickener with many industrial applications in food industry. Our aim was to estimate the ability of Xanthomonas campestris ATCC 13951 for the production of xanthan gum by using whey as a growth medium, a by-product of dairy industry. X. campestris ATCC 13951 has been studied in batch cultures using a complex medium for the determination of the optimal concentration of glucose, galactose and lactose. In addition, whey was used under various treatment procedures (de-proteinated, partially hydrolyzed by β-lactamase and partially hydrolyzed and de-proteinated) as culture medium, to study the production of xanthan in a 2 l bioreactor with constant stirring and aeration. A production of 28 g/l was obtained when partially hydrolysed β-lactamase was used, which proved to be one of the highest xanthan gum production reported so far. At the same time, an effort has been made for the control and selection of the most appropriate procedure for the preservation of the strain and its use as inoculant in batch cultures, without loss of its viability and its capability of xanthan gum production. The pre-treatment of whey (whey permeate medium hydrolyzed, WPH) was very important for the production of xanthan by the strain X. campestris ATCC 13951 during batch culture conditions in a 2 l bioreactor. Preservation methods such as lyophilization, cryopreservation at various glycerol solution and temperatures have been examined. The results indicated that the best preservation method for the producing strain X. campestris ATCC 13951 was the lyophilization. Taking into account that whey permeate is a low cost by-product of the dairy industry, the production of xanthan achieved under the studied conditions was considered very promising for industrial application.

The phosphoglucomutase gene from a wild type Fusarium oxysporum   strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45 °C but the enzyme became unstable at temperatures above 40 °C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co2+. The apparent Km for Co2+ was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn2+, Mg2+, Ni2+ and Ca2+ but to a lesser extent. The following kinetic constants were determined: vmax, 0.74 μmol mgprotein−1 min−1; kcat, 44.2 min−1; Km(G1P), 0.10 mM;Km(G1,6diP), 1.03 μM; kcat/Km(G1P), 443 mM−1 min−1 and kcat/Km(G1,6diP), 42,860 mM−1 min−1. The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.

In an effort to increase ethanol productivity during the consolidated bioprocessing (CBP) of lignocellulosics by Fusarium oxysporum, we attempted the constitutive homologous overexpression of one of the key process enzymes, namely an endo-xylanase. The endo-β-1,4-xylanase 2 gene was incorporated into the F. oxysporum genome under the regulation of the gpdA promoter of Aspergillus nidulans. The transformation was effected through Agrobacterium tumefaciens and resulted in 12 transformants, two of which were selected for further study due to their high extracellular xylanase activities under normally repressing conditions (glucose as sole carbon source). During natural induction conditions (growth on xylan) though, the extracellular enzyme levels of the transformants were only marginally higher (5–10%) compared to the wild type despite the significantly stronger xylanase 2 mRNA signals. SDS-PAGE verified enzyme assay results that there was no intracellular xylanase 2 accumulation in the transformants, suggesting the potential regulation in a post transcriptional or translational level. The fermentative performance of the transformants was evaluated and compared to that of the wild type in simple CBP systems using either corn cob or wheat bran as sole carbon sources. Both transformants produced approximately 60% more ethanol compared to the wild type on corn cob, while for wheat bran this picture was repeated for only one of them. This result is attributed to the high extracellular xylanase activities in the transformants’ fermentation broths that were maintained 2–2.5-fold higher compared to the wild type.

Four sampling sites were selected in the area of Velouhi mountain, Greece in order to screen for Pseudomonas syringae isolates with high ice nucleation activity from a ski resort environment. Bacterial isolates (n=147) were obtained from soil and phyllosphere samples. Seven isolates exhibited morphological, biochemical and physiological profile similar to P. syringae. Phylogenetic relationships of the seven isolates were determined by 16S rRNA gene sequencing. Two isolates were phylogenetically affiliated to P. syringae, three to P. viridiflava, one to P. avellanae, and one Pseudomonas strain could not be assigned to a known species. The seven isolates were examined for their ice-nucleation activity properties. Three out of the seven studied isolates exhibited ice nucleation activity from –4.67 to –4.35 ice nuclei per cell, values similar to those obtained from a known ice-nucleation protein producer P. syringae strain and therefore could be used for the production of artificial snow in ski resort areas with short snow periods.

Basic characteristics of on-line extractive fermentation of organic acids were examined using a general model for the integrated process in order to illustrate the effects of various process parameters and operational modes on system performance. The strong pH dependence of both the fermentation kinetics and the extraction efficiency has been outlined and taken into account by inclusion of a model equation which predicts the pH profile. Simulation results using data from butyric acid fermentations show that our complete model system is adequate to evaluate different operational modes of the process, including simple batch fermentation, batch or fed-batch fermentation with extractive recycle, and continuous fermentation with extractive recycle. In the case where a second undesired acidic byproduct is also produced, our model predictions suggest that on-line extractive fermentation using a suitable solvent results in an effective gross separation of the two acids.

One of the most important considerations in designing an integrated process for the production of organic acids is the strong pH dependence of the process parameters. The release of the acidic products during the fermentation alters the pH of the broth and subsequently affects the kinetics of cell growth and product formation. In addition, some parameters (such as the distribution coefficient of the products during extraction, their solubility constants during precipitation, etc.) in the subsequent separation processes following fermentation are also strong functions of pH. A pH profile predicting model for organic acids fermentation could be used in combination with specific separation process equations, in order to perform simulations concerning the feasibility of the whole process or the evaluation of the system's performance. In this work, such a general pH profile predicting model has been developed. The model requires knowledge of the fermentation medium composition and the kinetics of cell growth and product formation. The model has been verified for (but by no means restricted to) the case of butyric acid fermentation.