ALBERT

Description: In this study, the influence of twenty different single (i.e. 19 amino acids and ammonium sulphate) and two multiple nitrogen sources (N-sources) on growth and fermentation (i.e. glucose consumption and ethanol production) performance of Saccharomyces cerevisiae and of four wine-related non- Saccharomyces yeast species ( Lachancea thermotolerans , Metschnikowia pulcherrima , Hanseniaspora uvarum and Torulaspora delbrueckii ) was investigated during alcoholic fermentation. Briefly, the N-sources with beneficial effects on all performance parameters (or for the majority of them) for each yeast species were alanine, arginine, asparagine, aspartic acid, glutamine, isoleucine, ammonium sulphate, serine, valine and mixtures of 19 amino acids and of 19 amino acids plus ammonium sulphate (for S. cerevisiae ), serine (for L. thermotolerans ), alanine (for H. uvarum ), alanine and asparagine (for M. pulcherrima ), arginine, asparagine, glutamine, isoleucine and mixture of 19 amino acids (for T. delbrueckii ). Furthermore, our results showed a clear positive effect of complex mixtures of N-sources on S. cerevisiae and on T. delbrueckii (although to a lesser extent) as to all performance parameters studied, whereas for L. thermotolerans , H. uvarum and M. pulcherrima , single amino acids affected growth and fermentation performance to the same extent as the mixtures. Moreover, we found groups of N-sources with similar effects on the growth and/or fermentation performance of two or more yeast species. Finally, the influences of N-sources observed for T. delbrueckii and H. uvarum resembled those of S. cerevisiae the most and the least, respectively. Overall, this work contributes to an improved understanding of how different N-sources affect growth, glucose consumption and ethanol production of wine-related yeast species under oxygen-limited conditions, which, in turn, may be used to, e.g. optimize growth and fermentation performance of the given yeast upon N-source supplementation during wine fermentations.

Description: Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611 bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA Ser gene) via a 56-bp core sequence within the attP / attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa . The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa .

Description: In this work, Nannochloropsis salina was cultivated in a continuous-flow flat-plate photobioreactor, working at different residence times and irradiations to study the effect of the specific light supply rate on biomass productivity and photosynthetic efficiency. Changes in residence times lead to different steady-state cell concentrations and specific growth rates. We observed that cultures at steady concentration were exposed to different values of light intensity per cell. This specific light supply rate was shown to affect the photosynthetic status of the cells, monitored by fluorescence measurements. High specific light supply rate can lead to saturation and photoinhibition phenomena if the biomass concentration is not optimized for the selected operating conditions. Energy balances were applied to quantify the biomass growth yield and maintenance requirements in N. salina cells.

Description: An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis ( Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor ) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was 〉100-fold lower compared to strains expressing Zm gfor . Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l −1 and 11.5 g D-xylitol l −1 from 26 g D-xylose l −1 ), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l −1 ). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor , but decreased xylitol production in the strain expressing Zm gfor . In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.

Description: We have previously shown that overexpression of the human tumor suppressor protein P53 causes cell death of the yeast Saccharomyces cerevisiae . P53 overproduction led to transcriptional downregulation of some yeast genes, such as the TRX1/2 thioredoxin system, which plays a key role in cell protection against various oxidative stresses induced by reactive oxygen species (ROS). In the present work, the impact of TRX2 overexpression on apoptosis mediated by p53 overexpression in yeast is investigated. In yeast cells expressing P53 under an inducible promoter together with TRX2 under a strong constitutive promoter, we showed that Tr2p overproduction reduced the apoptotic effect exerted by P53 and increased the viability of the P53-overproducing cells. Furthermore, measurements of ROS amounts by flow cytometry and fluorescence microscopy indicated that the TRX2 protein acted probably through its increased detoxifying activity on the P53-generated ROS. The steady-state level and activity of P53 were not affected by TRX2 overexpression, as shown by western blotting and functional analysis of separated alleles in yeast (FASAY), respectively. The growth inhibitory effect of P53 was partially reversed by the antioxidant N -acetylcysteine. Our data strengthen the idea that overexpression of a single gene ( trx2 ) decreases the p53 -mediated cell death by decreasing ROS accumulation.

Description: The moving bed biofilm reactor-membrane bioreactor (MBBR-MBR) is a novel solution to conventional activated sludge processes and membrane bioreactors. In this study, a pure MBBR-MBR was studied. The pure MBBR-MBR mainly had attached biomass. The bioreactor operated with a hydraulic retention time (HRT) of 9.5 h. The kinetic parameters for heterotrophic and autotrophic biomasses, mainly nitrite-oxidizing bacteria (NOB), were evaluated. The analysis of the bacterial community structure of the ammonium-oxidizing bacteria (AOB), NOB, and denitrifying bacteria (DeNB) from the pure MBBR-MBR was carried out by means of pyrosequencing to detect and quantify the contribution of the nitrifying and denitrifying bacteria in the total bacterial community. The relative abundance of AOB, NOB, and DeNB were 5, 1, and 3 %, respectively, in the mixed liquor suspended solids (MLSS), and these percentages were 18, 5, and 2 %, respectively, in the biofilm density (BD) attached to carriers. The pure MBBR-MBR had a high efficiency of total nitrogen (TN) removal of 71.81 ± 16.04 %, which could reside in the different bacterial assemblages in the fixed biofilm on the carriers. In this regard, the kinetic parameters for autotrophic biomass had values of Y A  = 2.3465 mg O 2  mg N −1 , μ m, A  = 0.7169 h −1 , and K NH  = 2.0748 mg N L −1 .

Description: Slow sand filtration (SSF) is an effective low-tech water treatment method for pathogen and particle removal. Yet despite its application for centuries, it has been uncertain to which extent pathogenic microbes are removed by mechanical filtration or due to ecological interactions such as grazing and competition for nutrients. In this study, we quantified the removal of bacterial faecal indicators, Escherichia coli and Enterococcus faecalis , from secondary effluent of a wastewater treatment plant and analysed the microbial community composition in compartments of laboratory model SSF columns. The columns were packed with different sand grain sizes and eliminated 1.6–2.3 log units of faecal indicators, which translated into effluents of bathing water quality according to the EU directive (〈500 colony forming units of E. coli per 100 ml) for columns with small grain size. Most of that removal occurred in the upper filter area, the Schmutzdecke . Within that same zone, total bacterial numbers increased however, thus suggesting a specific elimination of the faecal indicators. The analysis of the microbial communities also revealed that some taxa were removed more from the wastewater than others. These results accentuate the contribution of biological mechanisms to water purification in SSF.

Description: Pseudoalteromonas is widespread in various marine environments, and most strains can affect invertebrate larval settlement and metamorphosis by forming biofilms. However, the impact and the molecular basis of population diversification occurring in Pseudoalteromonas biofilms are poorly understood. Here, we show that morphological diversification is prevalent in Pseudoalteromonas species during biofilm formation. Two types of genetic variants, wrinkled (frequency of 12 ± 5 %) and translucent (frequency of 5 ± 3 %), were found in Pseudoalteromonas lipolytica biofilms. The inducing activities of biofilms formed by the two variants on larval settlement and metamorphosis of the mussel Mytilus coruscus were significantly decreased, suggesting strong antifouling activities. Using whole-genome re-sequencing combined with genetic manipulation, two genes were identified to be responsible for the morphology alternations. A nonsense mutation in AT00 _ 08765 led to a wrinkled morphology due to the overproduction of cellulose, whereas a point mutation in AT00 _ 17125 led to a translucent morphology via a reduction in capsular polysaccharide production. Taken together, the results suggest that the microbial behavior on larval settlement and metamorphosis in marine environment could be affected by the self-generated variants generated during the formation of marine biofilms, thereby rendering potential application in biocontrol of marine biofouling.

Description: This study used an artificial enrichment microbial consortium to examine the effects of different substrate conditions on microbial diversity, composition, and function (e.g., zinc leaching efficiency) through adding pyrite (SP group), chalcopyrite (SC group), or both (SPC group) in sphalerite bioleaching systems. 16S rRNA gene sequencing analysis showed that microbial community structures and compositions dramatically changed with additions of pyrite or chalcopyrite during the sphalerite bioleaching process. Shannon diversity index showed a significantly increase in the SP (1.460), SC (1.476), and SPC (1.341) groups compared with control (sphalerite group, 0.624) on day 30, meanwhile, zinc leaching efficiencies were enhanced by about 13.4, 2.9, and 13.2 %, respectively. Also, additions of pyrite or chalcopyrite could increase electric potential (ORP) and the concentrations of Fe 3+ and H + , which were the main factors shaping microbial community structures by Mantel test analysis. Linear regression analysis showed that ORP, Fe 3+ concentration, and pH were significantly correlated to zinc leaching efficiency and microbial diversity. In addition, we found that leaching efficiency showed a positive and significant relationship with microbial diversity. In conclusion, our results showed that the complicated substrates could significantly enhance microbial diversity and activity of function.

Description: A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli . Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria , Bacteroidetes , Acidobateria , Firmicutes , Verrucomicrobia , Chloroflexi , Spirochaetes , Thermotogae , Armatimonadetes , and Planctomycetes . Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p -nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p -nitrophenol-α-( l )-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., 〉30 % methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.