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Concerning the potential application of the optically active isomer (R,R)-2,3-butanediol, and its production by a non-pathogenic bacterium Paenibacillus polymyxa ATCC 842, the present study evaluated the use of a commercial crude yeast extract Nucel®, as an organic nitrogen and vitamin source, at different medium composition and two airflows (0.2 or 0.5 vvm). The medium formulated (M4) with crude yeast extract carried out with the airflow of 0.2 vvm (experiment R6) allowed for a reduction in the cultivation time and kept the dissolved oxygen values at low levels until the total glucose consumption. Thus, the experiment R6 led to a fermentation yield of 41% superior when compared to the standard medium (experiment R1), which was conducted at airflow of 0.5 vvm. The maximum specific growth rate at R6 (0.42 h-1) was lower than R1 (0.60 h-1), however, the final cell concentration was not affected. Moreover, this condition (medium formulated-M4 and low airflow-0.2 vvm) was a great alternative to produce (R,R)-2,3-BD at fed-batch mode, resulting in 30 g.L-1 of the isomer at 24 h of cultivation, representing the main product in the broth (77%) and with a fermentation yield of 80%. These results showed that both medium composition and oxygen supply have an important role to produce 2,3-BD by P. polymyxa.

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The genomes of two Penicillium strains were sequenced and studied in this study: strain 2HH was isolated from the digestive tract of Anobium punctatum beetle larva in 1979 and the cellulase hypersecretory strain S1M29, derived from strain 2HH by a long-term mutagenesis process. With these data, the strains were reclassified and insight is obtained on molecular features related to cellulase hyperproduction and the albino phenotype of the mutant. Both strains were previously identified as Penicillium echinulatum and this investigation indicated that these should be reclassified. Phylogenetic and phenotype data showed that these strains represent a new Penicillium species in series Oxalica, for which the name Penicillium ucsense is proposed here. Six additional strains (SFC101850, SFCP10873, SFCP10886, SFCP10931, SFCP10932 and SFCP10933) collected from the marine environment in the Republic of Korea were also classified as this species, indicating a worldwide distribution of this new taxon. Compared to the closely related strain Penicillium oxalicum 114-2, the composition of cell wall-associated proteins of P. ucsense 2HH shows five fewer chitinases, considerable differences in the number of proteins related to ß-D-glucan metabolism. The genomic comparison of 2HH and S1M29 highlighted single amino-acid substitutions in two major proteins (BGL2 and FlbA) that can be associated with the hyperproduction of cellulases. The study of melanin pathways shows that the S1M29 albino phenotype resulted from a single amino-acid substitution in the enzyme ALB1, a precursor of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Our study provides important knowledge towards understanding species distribution, molecular mechanisms, melanin production and cell wall biosynthesis of this new Penicillium species.

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Penicillium echinulatum 2HH is an ascomycete well known for its production of cellulolytic enzymes. Understanding lignocellulolytic and sugar uptake systems is essential to obtain efficient fungi strains for the production of bioethanol. In this study we performed a genome-wide functional annotation of carbohydrate-active enzymes and sugar transporters involved in the lignocellulolytic system of P. echinulatum 2HH and S1M29 strains (wildtype and mutant, respectively) and eleven related fungi. Additionally, signal peptide and orthology prediction were carried out. We encountered a diverse assortment of cellulolytic enzymes in P. echinulatum, especially in terms of ß-glucosidases and endoglucanases. Other enzymes required for the breakdown of cellulosic biomass were also found, including cellobiohydrolases, lytic cellulose monooxygenases and cellobiose dehydrogenases. The S1M29 mutant, which is known to produce an increased cellulase activity, and the 2HH wild type strain of P. echinulatum did not show significant differences between their enzymatic repertoire. Nevertheless, we unveiled an amino acid substitution for a predicted intracellular ß-glucosidase of the mutant, which might contribute to hyperexpression of cellulases through a cellodextrin induction pathway. Most of the P. echinulatum enzymes presented orthologs in P. oxalicum 114-2, supporting the presence of highly similar cellulolytic mechanisms and a close phylogenetic relationship between these fungi. A phylogenetic analysis of intracellular ß-glucosidases and sugar transporters allowed us to identify several proteins potentially involved in the accumulation of intracellular cellodextrins. These may prove valuable targets in the genetic engineering of P. echinulatum focused on industrial cellulases production. Our study marks an important step in characterizing and understanding the molecular mechanisms employed by P. echinulatum in the enzymatic hydrolysis of lignocellulosic biomass.

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Penicillium echinulatum 2HH and Penicillium oxalicum 114-2 are well-known cellulase fungal producers. However, few studies addressing global mechanisms for gene regulation of these two important organisms are available so far. A recent finding that the 2HH wild-type is closely related to P. oxalicum leads to a combined study of these two species. Firstly, we provide a global gene regulatory network for P. echinulatum 2HH and P. oxalicum 114-2, based on TF-TG orthology relationships, considering three related species with well-known regulatory interactions combined with TFBSs prediction. The network was then analyzed in terms of topology, identifying TFs as hubs, and modules. Based on this approach, we explore numerous identified modules, such as the expression of cellulolytic and xylanolytic systems, where XlnR plays a key role in positive regulation of the xylanolytic system. It also regulates positively the cellulolytic system by acting indirectly through the cellodextrin induction system. This remarkable finding suggests that the XlnR-dependent cellulolytic and xylanolytic regulatory systems are probably conserved in both P. echinulatum and P. oxalicum. Finally, we explore the functional congruency on the genes clustered in terms of communities, where the genes related to cellular nitrogen, compound metabolic process and macromolecule metabolic process were the most abundant. Therefore, our approach allows us to confer a degree of accuracy regarding the existence of each inferred interaction.

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Abstract (1) Background: Oxygen supply is an important parameter to be considered in submerged cultures. This study evaluated the influence of different conditions for dissolved oxygen (DO) concentration on laccases activities and growth of Pleurotus sajor-caju PS-2001 in submerged process in stirred-tank bioreactor. (2) Methods: Initially, three different conditions were tested: uncontrolled DO and minimum levels of 30% and 80% of saturation, with the pH controlled between 4.5 and 7.0. (3) Results: Best results were observed at 30% DO (26 U mL-1 of laccases at 96 h), whereas higher mycelial biomass was observed at 30% and 80% DO (above 4.5 g L-1). Four different conditions of DO (uncontrolled, 10%, 30% and 50% of saturation) were tested at pH 6.5, with higher laccases activity (80 U mL-1 at 66 h) and lower mycelial growth (1.36 g L-1 at 90 h) being achieved with DO of 30%. In this test, the highest values for volumetric productivity and specific yield factor were determined. Under the different pH conditions tested, the production of laccases is favoured at DO concentration of 30% of saturation, while superior DO levels favours fungal growth. (4) Conclusion: The results indicate that dissolved oxygen concentration is a critical factor for the culture of P. sajor-caju PS-2001 and has important effects not only on laccases production but also on fungal growth.

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(1) Background: In this study, the effects of different pH values ​​(2.4, 3.2, 4.4 and 5.0), temperatures (30, 35, 40, 45 and 50°C) and agitation (100 rpm) on the enzymatic decolourisation of twenty-two dyes belonging to the chromophore groups anthraquinone, azo and triphenylmethane were assessed. (2) Methods: In all conditions, it was used a crude enzyme broth containing 30 U mL-1 laccases produced by Pleurotus sajor-caju PS-2001 in submerged process. (3) Results: Regarding the effects of pH values, the best results were obtained at pH 3.2 and 30°C, in which bleaching was observed for all dyes evaluated. In assays conducted at different temperatures, highest levels of decolourisation were observed at 35°C and pH 3.2 for nineteen of the dyes assessed. Thirteen dyes presented colour reduction exceeding 50% after the enzymatic treatment, including all acid and all disperse dyes evaluated. The reciprocal agitation of 100 rpm promoted negative effect on decolourisation. (4) Conclusion: From the results achieved, one can conclude that the laccase-containing preparation of P. sajor-caju PS-2001 has potential for the decolourisation of some dyes widely used in different industrial sectors, especially in the textile industry, and therefore could be used in future strategies for the biotreatment of coloured wastes.

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The potential to produce ethanol and xylitol from xylose by the macro basidiomycete Trametes membranacea was evaluated. All strains studied showed ethanol and xylitol production. The highest ethanol production of xylose was obtained by T. membranacea strain TM158/10 with 5.65 ± 0.21 g/L at pH 4 and 28 °C with 288 h of fermentation and 5.59 ± 0.05 g/L ethanol concentration at pH 5 and 24 °C with 360 h of fermentation. When the conversion was carried out using sugars generated from enzymatic hydrolysis of sugarcane bagasse, there were higher yields from 74 to 15% for ethanol and xylitol, respectively. Although the ethanol and xylitol production need to be optimized, this study showed for the first time the possibility of using T. membranacea for the simultaneous xylitol and ethanol production from pentose sugars, allowing for the possibility of using all released sugars during the hydrolysis of lignocelluloses.

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The performance of two lignocellulosic biomasses was studied in high-pressure carbon dioxide/water pre-treatment. Sugarcane bagasse and elephant grass were used to produce C5-sugars from hemicellulose and, simultaneously, to promote cellulose digestibility for enzymatic saccharification. Different pre-treatment conditions, with combined severity factor ranging from -1.17 to -0.04, were evaluated and maximal total xylan to xylose yields of 59.2wt.% (34.4wt.% xylooligomers) and 46.4wt.% (34.9wt.% xylooligomers) were attained for sugarcane bagasse and elephant grass, respectively. Furthermore, pre-treated biomasses were highly digestible, with glucan to glucose yields of 77.2mol% and 72.4mol% for sugarcane bagasse and elephant grass, respectively. High-pressure carbon dioxide/water pre-treatment provides high total C5-sugars and glucose recovery from both lignocellulosic biomasses; however it is highly influenced by composition and intrinsic features of each biomass. The obtained results confirm this approach as an effective and greener alternative to conventional pre-treatment processes.

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This study evaluated the potential use of elephant grass biomass, a highly productive species, for cellulase and xylanase production by the cellulolytic mutant Penicillium echinulatum 9A02S1 in submerged cultivation, using untreated biomass, biomass pretreated with different concentrations of NaOH, H2SO4 or NH4OH, or biomass pretreated with H2O at 121 °C. For filter paper activity, all cultivation carried out with pretreated elephant grass under the evaluated conditions showed superior activity when compared with the control (untreated elephant grass). The activities of endoglucanases and ß-glucosidases were higher in the cultivation prepared from pretreated samples than the control made with cellulose (Celuflok(®)). Without pretreatment, elephant grass can be used for xylanase production, enabling similar activities to those obtained in the cultivation with cellulose, reducing the enzyme production cost. These results indicate that the pretreatment of elephant grass, especially when pretreated with H2SO4, may be used as a partial or total replacement for cellulose to cellulase production, and untreated elephant grass may be used for xylanase production.

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The enzymatic hydrolysis of Pennisetum purpureum (elephant grass) was evaluated at high total solid levels (from 4% to 20% (w/v)) in a concomitant ball milling treatment in a rotating hydrolysis reactor (RHR). The greatest glucose yield was 20.17% when 4% (w/v) untreated biomass was employed. When sugars obtained from enzymatic hydrolysis were submitted to fermentation with Saccharomyces cerevisiae, the greatest ethanol yield was 22.61% when 4% (w/v) untreated biomass was employed; however, the highest glucose concentration (12.47g/L) was obtaining using 20% (w/v) solids and highest ethanol concentration (6.1g/L) was obtained using 16% (w/v) solids. When elephant grass was hydrolyzed in the rotating hydrolysis reactor, ethanol production was about double that was produced when the biomass was hydrolyzed in a static reactor (SR). These data indicate that it is possible to produce ethanol from elephant grass when milling treatment and enzymatic hydrolysis are performed at the same time.

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