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.

Description: Feruloyl or ferulic acid esterase (Fae, EC 3.1.1.73) catalyzes the hydrolysis of ester bonds between polysaccharides and phenolic acid compounds in xylan side chain. In this study, the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii was increased by iterative saturation mutagenesis (ISM). Two amino acids, Ser33 and Asn92, were selected for saturation mutagenesis according to the B-factors analyzed by B-FITTER software and ΔΔG values predicted by PoPMuSiC algorithm. After screening the saturation mutagenesis libraries constructed in Pichia pastoris , 15 promising variants were obtained. The best variant S33E/N92-4 (S33E/N92R) produced a T m value of 44.5 °C, the half-lives ( t 1/2 ) of 35 and 198 min at 55 and 50 °C, respectively, corresponding to a 4.7 °C, 2.33- and 3.96-fold improvement compared to the wild type. Additionally, the best S33 variant S33-6 (S33E) was thermostable at 50 °C with a t 1/2 of 82 min, which was 32 min longer than that of the wild type. All the screened S33E/N92 variants were more thermostable than the best S33 variant S33-6 (S33E). This work would contribute to the further studies on higher thermostability modification of type A feruloyl esterases, especially those from fungi. The thermostable feruloyl esterase variants were expected to be potential candidates for industrial application in prompting the enzymic degradation of plant biomass materials at elevated temperatures.

Description: Riemerella anatipestifer infection causes high mortality for ducks which results in major economic losses in the duck industry. In this study, we identified a mutant strain RA-M1 by Tn 4351 transposon mutagenesis, in which the M949_1603 gene encoding glycosyl transferase was inactivated. PCR analysis revealed that M949_1603 gene is specifically existed in R. anatipestifer serotype 1 strains. RA-M1 presented no reactivity to the anti-lipopolysaccharide (LPS) MAb in an indirect ELISA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting demonstrated that RA-M1 LPS had a deficiency in ladder-like binding pattern to rabbit antiserum against R. anatipestifer serotype 1 strain CH3, indicating that the O-antigen structure of RA-M1 was changed. RA-M1 showed significant attenuated virulence in ducks and higher sensitivity to normal duck serum, compared with its parent strain CH3. Furthermore, cross-protection of RA-M1 for R. anatipestifer serotypes 1, 2, and 10 strains was evaluated. Ducks that received two immunizations with inactivated RA-M1 vaccine were 100 % protected from challenge with R. anatipestifer serotype 1 strain WJ4, serotype 2 strain Yb2, and serotype 10 strain HXb2. No changes were observed in the liver, heart, or spleen samples from the protected ducks during autopsy and histological examination. Furthermore, vaccination generated high antibody titers of 1:12,800 against serotypes 1, 2, and 10 strains and enhanced production of interleukin 2 (IL-2) and IL-4 in ducks. These results suggested that M949_1603 gene is associated with serotype 1 O-antigen biosynthesis, and mutant RA-M1 could be used as a novel cross-protection vaccine candidate to protect ducks against R. anatipestifer infection.

Description: Saccharopolyspora spinosa can produce spinosad as a major secondary metabolite, which is an environmentally friendly agent for insect control. Cobalamin-independent methionine synthase (MetE) is an important enzyme in methionine biosynthesis, and this enzyme is probably closely related to spinosad production. In this study, its corresponding gene metE was inactivated, which resulted in a rapid growth and glucose utilisation rate and almost loss of spinosad production. A label-free quantitative proteomics-based approach was employed to obtain insights into the mechanism by which the metabolic network adapts to the absence of MetE. A total of 1440 proteins were detected from wild-type and Δ metE mutant strains at three time points: stationary phase of Δ metE mutant strain (S1 Δ metE , 67 h), first stationary phase of wild-type strain (S1 WT , 67 h) and second stationary phase of wild-type strain (S2 WT , 100 h). Protein expression patterns were determined using an exponentially modified protein abundance index (emPAI) and analysed by comparing S1 Δ metE /S1 WT and S1 Δ metE /S2 WT . Results showed that differentially expressed enzymes were mainly involved in primary metabolism and genetic information processing. This study demonstrated that the role of MetE is not restricted to methionine biosynthesis but rather is involved in global metabolic regulation in S. spinosa .

Description: Chlorella vulgaris encapsulated in alginate beads were added into a bioreactor treating synthetic wastewater using Pseudomonas putida . A symbiotic CO 2 /O 2 gas exchange was established between the two microorganisms for photosynthetic aeration of wastewater. During batch operation, glucose removal efficiency in the bioreactor improved from 50 % in 12 h without aeration to 100 % in 6 h, when the bioreactor was aerated photosynthetically. During continuous operation, the bioreactor was operated at a low hydraulic retention time of 3.3 h at feed concentrations of 250 and 500 mg/L glucose. The removal efficiency at 500 mg/L increased from 73 % without aeration to 100 % in the presence of immobilized microalgae. The initial microalgae concentration was critical to achieve adequate aeration, and the removal rate increased with increasing microalgae concentration. The highest removal rate of 142 mg/L-h glucose was achieved at an initial microalgae concentration of 190 mg/L. Quantification of microalgae growth in the alginate beads indicated an exponential growth during symbiosis, indicating that the bioreactor performance was limited by oxygen production rates. Under symbiotic conditions, the chlorophyll content of the immobilized microalgae increased by more than 30 %. These results indicate that immobilized microalgae in symbiosis with heterotrophic bacteria are promising in wastewater aeration.

Description: The microbiological production of 2,3-butanediol (2,3-BDO) has attracted considerable attention as an alternative way to produce high-value chemicals from renewable sources. Among the number of 2,3-BDO-producing microorganisms, Klebsiella pneumoniae has been studied most extensively and is known to produce large quantity of 2,3-BDO from a range of substrates. On the other hand, the pathogenic characteristics of the bacteria have limited its industrial applications. In this study, two major virulence traits, outer core LPS and fimbriae, were removed through homologous recombination from 2,3-BDO-producing K. pneumoniae 2242 to expand its uses to the industrial scale. The K. pneumoniae 2242 ∆wabG mutant strain was found to have an impaired capsule, which significantly reduced its ability to bind to the mucous layer and evade the phagocytic activity of macrophage. The association with the human ileocecal epithelial cell, HCT-8, and the bladder epithelial cell, T-24, was also reduced dramatically in the K. pneumoniae 2242 ∆fimA mutant strain that was devoid of fimbriae. However, the growth rate and production yield for 2,3-BDO were unaffected. The K. pneumoniae strains developed in this study, which are devoid of the major virulence factors, have a high potential for the efficient and sustainable production of 2,3-BDO.

Description: Various food proteins including, e.g. gluten, collagen and casein are rich in l -proline residues. Due to the cyclic structure of proline, these proteins are well protected from enzymatic degradation by typical digestive enzymes. Proline-specific peptidases (PsP) belong to different families of hydrolases acting on peptide bonds (EC 3.4.x.x). They occur in various organisms including bacteria, fungi, plants and insects. Based on their biochemical characteristics, PsP type enzymes are further grouped into different subclasses of which prolyl aminopeptidases (EC 3.4.11.5, PAP), prolyl carboxypeptidases (EC 3.4.17.16, PCP) and prolyl oligopeptidases/prolyl endopeptidases (EC 3.4.21.26, POP/PEP) are of major interest for applications in food biotechnology. This mini review summarises the biochemical assays employed for these subclasses of PsP and their structural properties and the reaction mechanisms. A special focus was set on PsP derived from fungi and insects and important industrial applications in the field of food biotechnology. The degradation of gluten and collagen as well as the hydrolysis of bitter peptides are discussed.

Description: The paper describes the preparation of new probiotic formulations based on chitosan-coated alginate microcapsules containing three different probiotic strains, Lactobacillus plantarum PBS067, Lactobacillus rhamnosus PBS070, and Bifidobacterium animalis subsp. lactis PBS075 taken individually and as a mixture of them. The effects of microencapsulation on the viability of the strains in conditions simulating the gastrointestinal tract and under industrial processes conditions were studied. In addition, an evaluation of their probiotic properties was also investigated by in vitro tests on the human intestinal cell line HT-29 to explore the effect of microencapsulation on health beneficial effect of the considered strains. Non-encapsulated cells were completely destroyed when exposed to simulated gastric juice and other stress conditions, while encapsulated cells exhibited a significantly higher resistance to artificial intestinal juice and heat and osmotic treatment. Moreover, in this study, the effect of the various microencapsulated probiotic strain formulations was compared with analogous formulations also containing the β-glucan Pleuran. The microencapsulation effectively protected the selected bacteria, as single strain and as a mixture of the three strains in both the formulations with and without Pleuran, from simulating gastrointestinal tract and industrial process conditions in delivering the viable cells without any significant adverse effect on their functionalities. The comparative study of the immunomodulatory properties of each single strain and the mixture of the three strains revealed a synergistic effect of the probiotic mixture, but no appreciable difference between the two kinds of formulations could be detected, as the effect of Pleuran is covered by the higher potential of the probiotic strains.

Description: Strategies for bioremediation of atrazine, a pesticide commonly polluting groundwater in low concentrations, were studied in two boreal nonagricultural soils. Atrazine was not mineralized in soil without bioremediation treatments. In biostimulation treatment with molasses, up to 52 % of atrazine was mineralized at 10 °C, even though the degradation gene copy numbers did not increase. Incubations with radioactively labeled atrazine followed by microautoradiographic analysis revealed that bioremediation strategies increased the relative proportion of active degraders from 0.3 up to 1.9 % of the total bacterial count. These results indicate that atrazine degradation might not solely be facilitated by atzA / trzN – atzB genes. In combined biostimulation treatment using citrate or molasses and augmentation with Pseudomonas citronellolis ADP or Arthrobacter aurescens strain TC1, up to 76 % of atrazine was mineralized at 30 °C, and the atrazine degradation gene numbers increased up to 10 7  copies g −1 soil. Clone libraries from passive samplers in groundwater monitoring wells revealed the presence of phylogenetic groups formerly shown to include atrazine degraders, and the presence of atrazine degradation genes atzA and atzB . These results show that the mineralization of low concentrations of atrazine in the groundwater zone at low temperatures is possible by bioremediation treatments.

Description: We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis . These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.

Description: Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae . Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

Description: Baculoviruses have a long history of safe use as specific, environmentally friendly insecticides that provide alternatives to chemical pesticides for controlling insect pests. However, their use has been limited by several factors, particularly their slow pathogenicity. In this study, we constructed a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) and an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that expressed an insect-specific cyto-insectotoxin (Cit1a) from the venom of the central Asian spider Lachesana tarabaevi . Cit1a is a comparatively long linear cytolytic molecule that contains a predicted α-helix structure composed of two short membrane-acting antimicrobial peptides (MAMPs) that are joined together in a “head-to-tail” shape. Cit1a fused to polyhedrin gene ( polh ) ( polh - cit1a ) was expressed in the nuclei as polyhedra in silkworm larvae, Bm5 and Sf9 cells. An early death of Bm5 and Sf9 cells by recombinant BmNPV/Polh-Cit1a and AcMNPV/Polh-Cit1a was observed compared with control viruses that lacked the toxin gene. The infected cells showed a loss of cytoplasm, membrane integrity, and structural changes, suggesting that recombinant baculovirus-infected cells were killed by the necrosis caused by Cit1a. In addition, the BmNPV/Polh-Cit1a showed a significant reduction in the median lethal time (LT 50 ) against silkworm larvae compared with those of control BmNPV that lacked the cit1a gene.

Description: Organic carbon, nitrogen, and sulfur are highly concentrated in municipal solid waste (MSW) landfill leachate, which usually frustrates conventional leachate treatment technologies from the perspective of energy costs. Therefore, the possibility of converting leachate to a new energy source via microbial fuel cell (MFC) technology has been examined recently. This paper summarizes the power output and energy recovery efficiency of the leachate-fed MFCs according to different feeding patterns, cell structures, and loading rates. Also, we assess potential energy-generating chemicals in leachate like nitrogen and sulfur compounds and propose alternative pathways, which may lift strict ratios between organic carbon and nitrogen content in conventional denitrification of leachate and are expected to achieve a higher voltage than traditional organic-oxygen based cells. Although currently power output of leachate-fed MFCs is limited, it seems well possible that dynamic characteristics of MSW leachates and microbial physiologies underlying some bio-electrochemically efficient activities (e.g., direct interspecies electron transfer) could be stimulated in MFC systems to improve the present status.

Description: Many filamentous fungi produce only conidia for dispersal and survival in vitro or in vivo. Here, we show that the developmental regulator WetA and the velvet protein VosA are not only required for conidial maturation but indispensable for conidiation in Beauveria bassiana , a filamentous entomopathogen. Deletion of wetA or vosA resulted in more than 90 % transcriptional depression of brlA and abaA , two activator genes in the central developmental pathway, during the critical period of conidiophore development and conidiation. Consequently, Δ wetA and Δ vosA strains lost 98 % in and 88 % of their conidiation capacities under optimal culture conditions, respectively. The conidia of Δ wetA showed more defective features than those of Δ vosA , including smaller size, lesser density, lower hydrophobicity, and impaired cell walls although intracellular trehalose content decreased more in the aging culture of Δ vosA than of Δ wetA . As a result, conidial sensitivity to cell wall perturbation was elevated in Δ wetA but unaffected in Δ vosA , which produced conidia more sensitive to the oxidant menadione and the wet-heat stress at 45 °C. Both deletion mutants showed similar defects in conidial tolerance to high osmolarity or UV-B irradiation but no change in conidial sensitivity to the other oxidant H 2 O 2 or the fungicide carbendazim. Moreover, Δ wetA lost more virulence to Galleria mellonella larvae than Δ vosA . All these phenotypical changes were restored by either wetA or vosA complementation. Taken together, WetA and VosA are indispensable for asexual development and contribute differentially to conidial quality and hence the biological control potential of B. bassiana against insect pests.

Description: The effect of chitosan with different molecular weights and other natural substances on dextransucrase (DSase) activity from a representative oral pathogen Streptococcus mutans was elucidated. Among other bioactive substances, amino-monosaccharides such as glucosamine, mannosamine, and galactosamine exerted the enzyme inhibitory activity over 95 % of DSase. The specified hydrolysates derived from the hydrolysis of high molecular weight chitosan (HMWC) designated to CTSN, CTSN-P, CTSN-B, and CTSN-S with different molecular weights ranging from 3 to 8 kDa showed the similar inhibitory activity toward DSase. Also, the hyaluronic acid (MW 8.9 kDa), sulfated chitin, and amino-monosaccharides demonstrated the significant activity, CTSN, CTSN-P, CTSN-B, and CTSN-S are of potent bioactive substances that can be prepared in the cheapest way compared with other molecules tested available for antibacterial agent useful for human oral health.

Description: Rifamycin is a broad-spectrum antimicrobial drug produced commercially by submerged fermentation where the yields are far less in comparison to its demand in human drug therapy. Addressing the need, sequential mutational strain improvement was carried using UV and EtBr that resulted in improved strain yielding rifamycin SV up to 4.32 g/L. Further optimization of six important fermentation factors was followed which include temperature, agitation, inoculum level, period of fermentation, inorganic nitrogen source and amino acids. For the first time, we report a maximum yield of 5.32 g/L of rifamycin SV. Among the amino acids, proline known for its slowest assimilation by Amycolatopsis mediterranei produced the highest improvement in antibiotic yields. Following mutational strain improvement and process optimization, a total of 3.8-fold increase in antibiotic titre was achieved. Following a conventional procedure of mutational strain improvement, highest yield of rifamycin SV was reported by optimizing submerged fermentation process.

Description: A new molecularly imprinted polymer (MIP), prepared by hispidin as the template molecule, was synthesized and applied as an adsorbent phase for solid phase extraction (SPE) to isolate and enrich hispidin from eight species of mushrooms. The optimization of synthesis and the adsorption behaviors of the MIPs were investigated in detail. In comparison with C 18 -SPE, MIP-SPE displayed high selectivity and good affinity for hispidin for extract of Phellinus igniarius. The antioxidant activity of the extracts after using the MIPs was evaluated by free radical scavenging activity, and inhibition of erythrocyte hemolysis, and lipid peroxidation. This developed method provided a rapid, selective, and effective approach for separation and enrichment of active compounds from the natural products.

Description: Vinyl chloride (VC), a known human carcinogen, is a common and persistent groundwater pollutant at many chlorinated solvent contaminated sites. The remediation of such sites is challenging because of the lack of knowledge on the microorganisms responsible for in situ VC degradation. To address this, the microorganisms involved in carbon assimilation from VC were investigated in a culture enriched from contaminated site groundwater using stable isotope probing (SIP) and high-throughput sequencing. The mixed culture was added to aerobic media, and these were amended with labeled ( 13 C-VC) or unlabeled VC ( 12 C-VC). The cultures were sacrificed on days 15, 32, and 45 for DNA extraction. DNA extracts and SIP ultracentrifugation fractions were subject to sequencing as well as quantitative PCR (qPCR) for a functional gene linked to VC-assimilation ( etnE ). The gene etnE encodes for epoxyalkane coenzyme M transferase, a critical enzyme in the pathway for VC degradation. The relative abundance of phylotypes was compared across ultracentrifugation fractions obtained from the 13 C-VC- and 12 C-VC-amended cultures. Four phylotypes were more abundant in the heavy fractions (those of greater buoyant density) from the 13 C-VC-amended cultures compared to those from the 12 C-VC-amended cultures, including Nocardioides , Brevundimonas , Tissierella , and Rhodoferax. Therefore, both a previously identified VC-assimilating genus ( Nocardioides ) and novel microorganisms were responsible for carbon uptake. Enrichment of etnE with time was observed in the heavy fractions, and etnE sequences illustrated that VC-assimilators harbor similar Nocardioides- like etnE . This research provides novel data on the microorganisms able to assimilate carbon from VC.

Description: As antibiotic resistance continues to spread globally, there is growing interest in the potential to limit the spread of antibiotic resistance genes (ARGs) from wastewater sources. In particular, operational conditions during sludge digestion may serve to discourage selection of resistant bacteria, reduce horizontal transfer of ARGs, and aid in hydrolysis of DNA. This study applied metagenomic analysis to examine the removal efficiency of ARGs through thermophilic and mesophilic anaerobic digestion using bench-scale reactors. Although the relative abundance of various ARGs shifted from influent to effluent sludge, there was no measureable change in the abundance of total ARGs or their diversity in either the thermophilic or mesophilic treatment. Among the 35 major ARG subtypes detected in feed sludge, substantial reductions (removal efficiency 〉90 %) of 8 and 13 ARGs were achieved by thermophilic and mesophilic digestion, respectively. However, resistance genes of aad A, mac B, and sul 1 were enriched during the thermophilic anaerobic digestion, while resistance genes of erythromycin esterase type I, sul 1, and tet M were enriched during the mesophilic anaerobic digestion. Efflux pump remained to be the major antibiotic resistance mechanism in sludge samples, but the portion of ARGs encoding resistance via target modification increased in the anaerobically digested sludge relative to the feed. Metagenomic analysis provided insight into the potential for anaerobic digestion to mitigate a broad array of ARGs.

Description: Yersinia pestis , an infectious bacterium that is a causative agent of plague, a disease which has been shown to be one of the most feared in history and which has caused millions of deaths. The capsule-like fraction 1 (F1) antigen expressed by Y. pestis is a known specific marker for the identification of the bacteria; therefore, the detection of F1 is important for Y. pestis recognition. In this study, a rapid, sensitive, and specific technique, the lateral flow assay (LFA), was successfully developed to detect Y. pestis by the recombinant F1 antigen. The assay that utilized an anti-F1 polyclonal antibody (Pab) to identify the bacteria was based on a double-antibody sandwich format on a nitrocellulose membrane. With the LFA method, 50 ng/ml of recombinant F1 protein and 10 5 CFU/mL of Y. pestis could be detected in less than 10 min. This assay also showed no cross-reaction with other Yersinia spp. or with some selected capsule-producing Enterobacteriaceae strains. Furthermore, detection of Y. pestis in simulated samples has been evaluated. The detection sensitivity of Y. pestis in various matrices was 10 5 CFU/mL, which was identical to that in PBS buffer. The results obtained suggest that LFA is an excellent tool for detection of Y. pestis contamination in an environment and hence can be used to monitor plague diseases when they emerge.

Description: Microbial production of biodiesel from renewable feedstock has attracted intensive attention. Biodiesel is known to be produced from short-chain alcohols and fatty acyl-CoAs through the expression of wax ester synthase/fatty acyl-CoA: diacylglycerol acyltransferase that catalyzes the esterification of short-chain alcohols and fatty acyl-CoAs. Here, we engineered Escherichia coli to produce various fatty alcohol acetate esters, which depend on the expression of Saccharomyces cerevisiae alcohol acetyltransferase ATF1 that catalyzes the esterification of fatty alcohols and acetyl-CoA. The fatty acid biosynthetic pathways generate fatty acyl-ACPs, fatty acyl-CoAs, or fatty acids, which can be converted to fatty alcohols by fatty acyl-CoA reductase, fatty acyl-ACP reductase, or carboxylic acid reductase, respectively. This study showed the biosynthesis of biodiesel from three fatty acid biosynthetic pathway intermediates.

Description: Epidermal growth factor (EGF) ameliorates stress and prevents incomplete gastrointestinal development in early-weaned piglets in commercial swine farming. This study aimed to further analyze the biological activities of intracellularly expressed EGF (IE-EGF), extracellularly expressed EGF (EE-EGF), and tagged EGF (T-EGF) from Saccharomyces cerevisiae in early-weaned pigs. In this study, we assigned 24 pigs to each of 5 groups that were provided a basic diet (the control group) or a diet supplemented with empty vector-expressing S. cerevisia e [the INVSc1(EV) group], T-EGF-expressing S. cerevisiae [the INVSc1-TE(−) group], EE-EGF-expressing S. cerevisiae [the INVSc1-EE(+) group], or IE-EGF-expressing S. cerevisiae [the INVSc1-IE(+) group]. All treatments were delivered at a dose of 60 μg EGF/kg body weight (BW) everyday. All the piglets were sacrificed after 21 day to determine their physio-biochemical indexes, immune functions, and intestinal development. In the piglet experiments, recombinant S. cerevisiae survived throughout the intestinal tract. The BW and intestinal development (e.g., mean villous height, crypt depth, villous height:crypt depth ratio (IVR), and total protein, DNA, and RNA contents) of the piglets were significantly enhanced in the INVSc1-IE(+) group compared with the animals in the INVSc1-EE(+) and INVSc1-TE(−) groups ( P  〈 0.05). In addition, increased proliferating cell nuclear antigen (PCNA) staining was observed in the piglets that received the INVSc1-IE(+) treatment (approximately 80 %) compared with those that received the INVSc1-TE(−) (approximately 70 %) and INVSc1-EE(+) treatments (approximately 70 %). The levels of lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (ALP), immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) were also significantly increased in the INVSc1-IE(+) group compared with the INVSc1-EE(+) and INVSc1-TE(−) groups ( P  〈 0.05). Furthermore, the proliferation of piglet enterocytes was also significantly stimulated by both IE-EGF and EE-EGF compared with T-EGF in vitro ( P  〈 0.05). Our data further demonstrate the previously reported hypothesis that IE-EGF is more suitable than EE-EGF or T-EGF for applications in early-weaned pigs.

Description: With the rapid development of molecular diagnostic techniques, there is a growing need for quality controls and standards with favorable properties to monitor the entire detection process. In this study, we describe a novel method to produce armored hepatitis B virus (HBV) and human papillomavirus (HPV) DNA for use in nucleic acid tests, which was confirmed to be stable, homogeneous, noninfectious, nuclease resistant, and safe for shipping. We demonstrated that MS2 bacteriophage could successfully package double-stranded DNA of 1.3-, 3-, 3.5-, and 6.5-kb length into viral capsids with high reassembly efficiency. This is the first application of RNA bacteriophage MS2 as a platform to encapsulate double-stranded DNA, forming virus-like particles (VLPs) which were indistinguishable from native MS2 capsids in size and morphology. Moreover, by analyzing the interaction mechanism of pac site and the MS2 coat protein (CP), we found that in addition to the recognized initiation signal TR-RNA, TR-DNA can also trigger spontaneous reassembly of CP dimers, providing a more convenient and feasible method of assembly. In conclusion, this straightforward and reliable manufacturing approach makes armored DNA an ideal control and standard for use in clinical laboratory tests and diagnostics, possessing prospects for broad application, especially providing a new platform for the production of quality controls for DNA viruses.

Description: Lipids are naturally derived products that offer an attractive, renewable alternative to petroleum-based hydrocarbons. While naturally produced long-chain fatty acids can replace some petroleum analogs, medium-chain fatty acid would more closely match the desired physical and chemical properties of currently employed petroleum products. In this study, we engineered Yarrowia lipolytica , an oleaginous yeast that naturally produces lipids at high titers, to produce medium-chain fatty acids. Five different acyl-acyl carrier protein (ACP) thioesterases with specificity for medium-chain acyl-ACP molecules were expressed in Y. lipolytica , resulting in formation of either decanoic or octanoic acid. These novel fatty acid products were found to comprise up to 40 % of the total cell lipids. Furthermore, the reduction in chain length resulted in a twofold increase in specific lipid productivity in these engineered strains. The medium-chain fatty acids were found to be incorporated into all lipid classes.

Description: Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM β-CD and 150 μM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that β-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that β-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that β-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus , and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids.

Description: Mechanisms of glutathione (GSH) over-accumulation in mutant Saccharomyces cerevisiae Y518 screened by ultraviolet and nitrosoguanidine-induced random mutagenesis were studied. Y518 accumulated higher levels of GSH and l -cysteine than its wild-type strain. RNA-Seq and pathway enrichment analysis indicated a difference in the expression of key genes involved in cysteine production, the GSH biosynthesis pathway, and antioxidation processes. GSH1 , MET17 , CYS4 , GPX2 , CTT1 , TRX2 , and SOD1 and the transcriptional activators SKN7 and YAP1 were up-regulated in the mutant. Moreover, Y518 showed a dysfunctional respiratory chain resulting from dramatically weakened activity of complex III and significant elevation of intracellular reactive oxygen species (ROS) levels. The supplementation of antimycin A in the culture of the parent strain showed equivalent changes of ROS and GSH level. This study indicates that defective complex III prompts abundant endogenic ROS generation, which triggers an oxidative stress response and upregulation of gene expression associated with GSH biosynthesis. This finding may be helpful for developing new strategies for GSH fermentation process optimization or metabolic engineering.

Description: Prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily are involved in the biosynthesis of secondary metabolites and contribute as modification enzymes significantly to structural diversity of natural products. They show usually broad specificity toward their aromatic substrates with regiospecific prenylations on aromatic rings. However, most members of this superfamily exhibit a high specificity toward their prenyl donors and usually accept exclusively dimethylallyl diphosphate (DMAPP). Recently, several indole prenyltransferases from this family were also demonstrated to accept unnatural DMAPP analogs such as methylallyl, 2-pentenyl and benzyl diphosphate for alkylation, or benzylation of the indole ring. Partial or complete shift of the substitution position was observed for these enzymes. In this study, we report the acceptance of these DMAPP analogs by two tyrosine O -prenyltransferases TyrPT from Aspergillus niger and SirD from Leptosphaeria maculans for alkylation or benzylation of tyrosine and derivatives. NMR and mass spectrometry (MS) analyses of nine isolated enzyme products confirmed the regiospecific O- or N -alkylation or benzylation at position C-4 of the aromatic ring, which is the same prenylation position of these enzymes in the presence of DMAPP.

Description: Isolates of Aspergillus species are able to produce a large number of secondary metabolites. The profiles of biosynthetic families of secondary metabolites are species specific, whereas individual secondary metabolite families can occur in other species, even those phylogenetically and ecologically unrelated to Aspergillus . Furthermore, there is a high degree of chemo-consistency from isolate to isolate in a species even though certain metabolite gene clusters are silenced in some isolates. Genome sequencing projects have shown that the diversity of secondary metabolites is much larger in each species than previously thought. The potential of finding even further new bioactive drug candidates in Aspergillus is evident, despite the fact that many secondary metabolites have already been structure elucidated and chemotaxonomic studies have shown that many new secondary metabolites have yet to be characterized. The genus Aspergillus is cladistically holophyletic but phenotypically polythetic and very diverse and is associated to quite different sexual states. Following the one fungus one name system, the genus Aspergillus is restricted to a holophyletic clade that include the morphologically different genera Aspergillus , Dichotomomyces , Phialosimplex , Polypaecilum and Cristaspora . Secondary metabolites common between the subgenera and sections of Aspergillus are surprisingly few, but many metabolites are common to a majority of species within the sections. We call small molecule extrolites in the same biosynthetic family isoextrolites. However, it appears that secondary metabolites from one Aspergillus section have analogous metabolites in other sections (here also called heteroisoextrolites). In this review, we give a genus-wide overview of secondary metabolite production in Aspergillus species. Extrolites appear to have evolved because of ecological challenges rather than being inherited from ancestral species, at least when comparing the species in the different sections of Aspergillus . Within the Aspergillus sections, secondary metabolite pathways seem to inherit from ancestral species, but the profiles of these secondary metabolites are shaped by the biotic and abiotic environment. We hypothesize that many new and unique section-specific small molecule extrolites in each of the Aspergillus will be discovered.

Description: Gene amplification using dihydrofolate reductase gene ( dhfr ) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3′-untranslated region (UTR) of endogenous dhfr . Thus, shRNAs were designed to target the 3′-UTR of endogenous dhfr , and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80 % in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity ( q EPO ) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr . This study reveals that an expression vector including shRNA that targets the 3′-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

Description: Biological activities of medicinal mushrooms have been attributed to β-(1→3),(1→6)-glucans that are present in the cell wall of fungi and some plants. Antitumor, immunomodulatory, antimicrobial, antinociception, antiinflammatory, prebiotic, antioxidant, and antidiabetic are some of different properties already described for β-(1→3),(1→6)-glucans. Immune activation systems, including specific β-glucan receptors like Dectin-1, complement (CR3), and Toll (TLR), have been identified to clarify these biological effects. The β-(1→3)-glucans are synthesized by β-(1→3)-glucan synthase (GLS), an enzyme belonging to the glucosyltransferase group, which has a catalytic unit (FKS) and another regulatory (RHO). The mechanisms for adding β-(1→6) branches to the non-reducing ends of the β-(1→3)-glucan chains are unclear until now. Due to the biological importance of β-(1→3),(1→6)-glucan, it is necessary to understand the biochemical and molecular mechanisms of its synthesis, both to optimize the production of bioactive compounds and to develop antifungal drugs that interrupt this process. Therefore, the aim of this review is to gather information about the potential of β-(1→3),(1→6)-glucans, their methods of isolation, purification, and chemical characterization, as well as how these biomolecules are synthesized by fungi and what studies involving biotechnology or molecular biology have contributed to this subject.

Description: Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.

Description: Numerous species of wild-grown mushrooms are among the most vulnerable organisms for contamination with radiocesium released from a radioactive fallout. A comparison was made on radiocesium as well as the natural gamma ray-emitting radionuclide ( 40 K) activity concentrations in the fruiting bodies of several valued edible Boletus mushrooms collected from the region of Europe and Yunnan Province in China. Data available for the first time for Boletus edulis collected in Yunnan, China, showed a very weak contamination with 137 Cs. Radiocesium concentration activity of B. edulis samples that were collected between 2011 and 2014 in Yunnan ranged from 5.2 ± 1.7 to 10 ± 1 Bq kg −1 dry matter for caps and from 4.7 ± 1.3 to 5.5 ± 1.0 Bq kg −1 dry matter for stipes. The mushrooms Boletus badius , B. edulis , Boletus impolitus , Boletus luridus , Boletus pinophilus , and Boletus reticulatus collected from the European locations between 1995 and 2010 showed two to four orders of magnitude greater radioactivity from 137 Cs compared to B. edulis from Yunnan. The nuclide 40 K in B. badius was equally distributed between the caps and stipes, while for B. edulis , B. impolitus , B. luridus , B. pinophilus , and B. reticulatus , the caps were richer, and for each mushroom, activity concentration seemed to be more or less species-specific.

Description: A novel TaqMan-based multiplex real-time PCR method combined with propidium monoazide (PMA) treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10 8  CFU/mL dead cells of both bacteria. The high specificity of this method was confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 10 1 –10 2  CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100 and 106 % for V. parahaemolyticus and L. monocytogenes , respectively. Furthermore, this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4 °C. In conclusion, this PMA TaqMan-based multiplex real-time PCR technique, where the whole procedure takes less than 5 h, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health.

Description: A hybrid airlift reactor was adopted to retain aerobic granules in the reactor successfully for continuous operation. It was found that aerobic granules maintained excellent physical structure stability in the continuous-flow reactor with reactor performance as good as batch operation. However, flocs appeared after batch operation was switched to continuous operation, and chemical oxygen demand (COD) in the wastewater was thus removed by co-existed granules and flocs in the reactor. Furthermore, excessive precipitation of CaCO 3 as needled shaped aragonite in the continuous aerobic granular sludge reactor was observed, which led to the further enhancement of settling ability of granules with sludge volume index (SVI) reduction from 32 to 2 ml g −1 but specific oxygen utilization rate (SOUR) decrease from 61 to 23 mg O 2  g −1 MLVSS h −1 . Thus, apart from the physical structure stability, bioactivity stability of granules should be also considered as an important parameter to evaluate the continuous operation of aerobic granular sludge. Furthermore, the decrease in granule polysaccharide content implied that protein was more important for aragonite precipitation. The excessive aragonite precipitation in the continuous-flow reactor could be due to the competition between flocs and granules. In addition, the degradation of polysaccharide in aerobic granules under a continuous-flow mode may also contribute to excessive aragonite precipitation.

Description: Venom, the mucus layer covering the body surface, ink glands, mammary glands, milk, and various animal secretory functions as both a physical and chemical defense barrier against bacteria and virus infections. Previously, several studies reported that l -amino acid oxidases (LAAOs) present in animal secretary fluids have strong antimicrobial activities and selective cytotoxic activities against Gram-positive and Gram-negative bacteria, various pathogenic bacteria, viruses, and parasite species. These LAAOs catalyze oxidative deamination of an l -amino acid substrate with the generation of hydrogen peroxide. The antibacterial activity of LAAOs is completely inhibited by catalase; thus, LAAOs kill bacteria by the hydrogen peroxide generated from the oxidation of l -amino acid substrates. This review focuses on the selective, specific, and local antibacterial actions of various LAAOs that may be used as novel therapeutic agents against infectious diseases. LAAOs that are suitable leads for combating multidrug-resistant bacterial infections are also studied.

Description: Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

Description: Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus . By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra( p -toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA - lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S . aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant ( gyrB ), as opposed to wild type, neither RecA protein level nor cell’s susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S . aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.

Description: Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18- rDNA2 - ura 3- P pgk - 5hsCT - rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb ( rDNA1 ) and 1.4 kb ( rDNA2 ), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene ( 5hsCT ) under the control of the promoter for phosphoglycerate kinase ( P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura 3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease Hpa I, a linear fragment, rDNA2 - ura 3- P pgk - 5hsCT - rDNA1 , was obtained and transformed into the △ ura3 mutant of S. cerevisiae by the lithium acetate method. The ura 3- P pgk - 5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS- 5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae . The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS- 5hsCT . The expression level reached 2.04 % of total proteins. S. cerevisiae YS- 5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS- 5hsCT /kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

Description: Since total petroleum hydrocarbons (TPH) are toxic and persistent in environments, studying the impact of oil contamination on microbial communities in different soils is vital to oil production engineering, effective soil management and pollution control. This study analyzed the impact of oil contamination on the structure, activity and function in carbon metabolism of microbial communities of Chernozem soil from Daqing oil field and Cinnamon soil from Huabei oil field through both culture-dependent techniques and a culture-independent technique—pyrosequencing. Results revealed that pristine microbial communities in these two soils presented disparate patterns, where Cinnamon soil showed higher abundance of alkane, (polycyclic aromatic hydrocarbons) PAHs and TPH degraders, number of cultivable microbes, bacterial richness, bacterial biodiversity, and stronger microbial activity and function in carbon metabolism than Chernozem soil. It suggested that complicated properties of microbes and soils resulted in the difference in soil microbial patterns. However, the changes of microbial communities caused by oil contamination were similar in respect of two dominant phenomena. Firstly, the microbial community structures were greatly changed, with higher abundance, higher bacterial biodiversity, occurrence of Candidate_division_BRC1 and TAO6 , disappearance of BD1-5 and Candidate_division_OD1 , dominance of Streptomyces , higher percentage of hydrocarbon-degrading groups, and lower percentage of nitrogen-transforming groups. Secondly, microbial activity and function in carbon metabolism were significantly enhanced. Based on the characteristics of microbial communities in the two soils, appropriate strategy for in situ bioremediation was provided for each oil field. This research underscored the usefulness of combination of culture-dependent techniques and next-generation sequencing techniques both to unravel the microbial patterns and understand the ecological impact of contamination.

Description: Hydrogen is a promising alternative as an energetic carrier and its production by dark fermentation from wastewater has been recently proposed, with special attention to crude glycerol as potential substrate. In this study, two different feeding strategies were evaluated for replacing the glucose substrate by glycerol substrate: a one-step strategy (glucose was replaced abruptly by glycerol) and a step-by-step strategy (progressive decrease of glucose concentration and increase of glycerol concentration from 0 to 5 g L −1 ), in a continuous stirred tank reactor (12 h of hydraulic retention time (HRT), pH 5.5, 35 °C). While the one-step strategy led to biomass washout and unsuccessful H 2 production, the step-by-step strategy was efficient for biomass adaptation, reaching acceptable hydrogen yields (0.4 ± 0.1 mol H2  mol −1 glycerol consumed ) around 33 % of the theoretical yield independently of the glycerol concentration. Microbial community structure was investigated by single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) fingerprinting techniques, targeting either the total community (16S ribosomal RNA (rRNA) gene) or the functional Clostridium population involved in H 2 production (hydA gene), as well as by 454 pyrosequencing of the total community. Multivariate analysis of fingerprinting and pyrosequencing results revealed the influence of the feeding strategy on the bacterial community structure and suggested the progressive structural adaptation of the community to increasing glycerol concentrations, through the emergence and selection of specific species, highly correlated to environmental parameters. Particularly, this work highlighted an interesting shift of dominant community members (putatively responsible of hydrogen production in the continuous stirred tank reactor (CSTR)) according to the gradient of glycerol proportion in the feed, from the family Veillonellaceae to the genera Prevotella and Clostridium sp., putatively responsible of hydrogen production in the CSTR.

Description: Fucosyl- N -acetylglucosamine disaccharides are important core structures that form part of human mucosal and milk glyco-complexes. We have previously shown that AlfB and AlfC α-L-fucosidases from Lactobacillus casei are able to synthesize fucosyl-α-1,3-- N -acetylglucosamine (Fuc-α1,3-GlcNAc) and fucosyl-α-1,6- N -acetylglucosamine (Fuc-α1,6-GlcNAc), respectively, in transglycosylation reactions. Here, these reactions were performed in a semipreparative scale, and the produced disaccharides were purified. The maximum yields obtained of Fuc-α1,3-GlcNAc and Fuc-α1,6-GlcNAc were 4.2 and 9.3 g/l, respectively. The purified fucosyl-disaccharides were then analyzed for their prebiotic effect in vitro using strains from the Lactobacillus casei / paracasei / rhamnosus group and from Bifidobacterium species. The results revealed that 6 out of 11  L. casei strains and 2 out of 6  L. rhamnosus strains tested were able to ferment Fuc-α1,3-GlcNAc, and L. casei BL87 and L. rhamnosus BL327 strains were also able to ferment Fuc-α1,6-GlcNAc. DNA hybridization experiments suggested that the metabolism of Fuc-α1,3-GlcNAc in those strains relies in an α-L-fucosidase homologous to AlfB. Bifidobacterium breve and Bibidobacterium pseudocatenolatum species also metabolized Fuc-α1,3-GlcNAc. Notably, L-fucose was excreted from all the Lactobacillus and Bifidobacterium strains fermenting fucosyl-disaccharides, except from strains L. rhamnosus BL358 and BL377, indicating that in these latest strains, L-fucose was catabolized. The fucosyl-disaccharides were also tested for their inhibitory potential of pathogen adhesion to human colon adenocarcinoma epithelial (HT29) cell line. Enteropathogenic Escherichia coli (EPEC) strains isolated from infantile gastroenteritis were used, and the results showed that both fucosyl-disaccharides inhibited adhesion to different extents of certain EPEC strains to HT29 cells in tissue culture.

Description: Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis ) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter .

Description: Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR® green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor , which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor . In order to demonstrate that this SYBR® green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor , environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods.

Description: Alphaproteobacterium strain Q-1 produces an extracellular multicopper oxidase (IOX), which catalyzes iodide (I – ) oxidation to form molecular iodine (I 2 ). In this study, the antimicrobial activity of the IOX/iodide system was determined. Both Gram-positive and Gram-negative bacteria tested were killed completely within 5 min by 50 mU mL –1 of IOX and 10 mM iodide. The sporicidal activity of the system was also tested and compared with a common iodophor, povidone-iodine (PVP-I). IOX (300 mU mL –1 ) killed Bacillus cereus , B. subtilis , and Geobacillus stearothermophilus spores with decimal reduction times of 2.58, 7.62, and 40.9 min, respectively. However, 0.1 % PVP-I killed these spores with much longer decimal reduction times of 5.46, 38.0, and 260 min, respectively. To evaluate the more superior sporicidal activity of the IOX system over PVP-I, the amount of free iodine (non-complexed I 2 ) was determined by an equilibrium dialysis technique. The IOX system included more than 40 mg L –1 of free iodine, while PVP-I included at most 25 mg L –1 free iodine. Our results suggest that the new enzyme-based antimicrobial system is effective against a wide variety of microorganisms and bacterial spores, and that its strong biocidal activity is due to its high free iodine content, which is probably maintained by re-oxidation of iodide released after oxidation of cell components by I 2 .

Description: Many different organic compounds may be converted by microbial biotransformation to high-value products for the chemical and pharmaceutical industries. This review summarizes the use of strains of Aspergillus niger , a well-known filamentous fungus used in numerous biotechnological processes, for biochemical transformations of organic compounds. The substrates transformed include monocyclic, bicyclic, and polycyclic aromatic hydrocarbons; azaarenes, epoxides, chlorinated hydrocarbons, and other aliphatic and aromatic compounds. The types of reactions performed by A. niger , although not unique to this species, are extremely diverse. They include hydroxylation, oxidation of various functional groups, reduction of double bonds, demethylation, sulfation, epoxide hydrolysis, dechlorination, ring cleavage, and conjugation. Some of the products may be useful as new investigational drugs or chemical intermediates.

Description: Nitrogen is an important nutrient in alcoholic fermentation because its starvation affects both fermentation kinetics and the formation of yeast metabolites. In most alcoholic fermentations, yeasts have to ferment in nitrogen-starved conditions, which requires modifications of cell functions to maintain a high sugar flux and enable cell survival for long periods in stressful conditions. In this review, we present an overview of our current understanding of the responses of the wine yeast Saccharomyces cerevisiae to variations of nitrogen availability. Adaptation to nitrogen starvation involves changes in the activity of signaling pathways such as target of rapamycin (TOR) and nitrogen catabolite repression (NCR), which are important for the remodeling of gene expression and the establishment of stress responses. Upon starvation, protein degradation pathways involving autophagy and the proteasome play a major role in nitrogen recycling and the adjustment of cellular activity. Recent progress in the understanding of the role of these mechanisms should enable advances in fermentation management and the design of novel targets for the selection or improvement of yeast strains.

Description: Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v ), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later.

Description: Levansucrase catalyzes three distinct reactions depending on the fructosyl acceptor molecule, including polymerization, transfructosylation, and hydrolysis. As a key biocatalyst in the synthesis of levan and levan-type fructooligosaccharides, levansucrase has been widely and intensively studied. Due to the promising physiological effects of levan and levan-type fructooligosaccharides, they exhibit great potential in food and pharmaceutical industries. Another important point that attracts much attention is the wide substrate specificity of levansucrase toward monosaccharides, disaccharides, and aromatic and alkyl alcohols, producing diverse sucrose analogues, hetero-oligosaccharides (especially lactosucrose), and interesting fructosides. The present article summarizes and discusses the recent applications of microbial levansucrase in detail.

Description: Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds.

Description: Ginseng is one of the most important plants in oriental medicine. The pharmacological effects of this medicinal herb are mostly correlated to the major bioactive triterpene saponin, called ginsenoside. Due to the long cultivation period of ginseng and increased ginsenoside level in aged root, we need to develop strategies to increase ginseng productivity in cell and tissue culture in a faster way. Elicitation is already considered to improve the yield of this valuable secondary metabolite; especially, different types, timings, and durations of elicitation could affect the ginsenoside production and heterogeneity. Activation of ginsenoside biosynthetic genes and ginsenoside accumulation mediated by elicitor-induced signaling molecules would be helpful for commercial production of individual ginsenosides. Jasmonic acid is the well-known signaling molecule which mainly involved in ginsenoside accumulation. Ca 2+ spiking and reactive oxygen species, nitric oxide, and ethylene production are other messengers which mediate production of ginsenoside. This review highlights the elicitation strategies for production of the ginsenoside based on the principle of putative signal transduction pathways.

Description: Effects of some key factors on hydrogenotrophic denitrification were investigated in lab-scale bioreactors. Results indicated that optimum biomass loading, pH value, temperature, nitrate loading, and C/N ratio in this study were optical density at 600 nm (OD 600 ) of 0.173, 6.0∼7.0, 35 °C, 105 mg L −1 , and 30, respectively. To obtain a better understanding of microbial community in the bioreactors, the 454-pyrosequencing technology was used to characterize the 16S ribosomal RNA (16S rRNA) gene of bacteria in selected samples Y1 and Y2, in which a total of 62,559 effective sequences (36,445 in Y1 and 26,114 in Y2) were obtained. The taxonomic complexities in two samples were compared at phylum/class/genus levels. In total, 41 bacterial phyla, 43 bacterial classes, and 312 genera were detected, where phylum Firmicutes , class Clostridia were most abundant. Genus Proteiniclasticum was predominant among the top 100 genera. This work aims to add some novel insights into hydrogenotrophic denitrification process and its microbial community structures in bioreactors.

Description: Nitrite-dependent anaerobic methane oxidation (n-damo) is a newly discovered bioprocess that reduces nitrite to dinitrogen with methane as electron donor, which has promising potential to remove nitrogen from wastewater. In this work, a lab-scale sequencing batch reactor (SBR) was operated for 609 days with methane as the sole external electron donor. In the SBR, nitrite in synthetic wastewater was removed continuously; the final volumetric nitrogen removal rate was 12.22±0.02 mg N L −1  day −1 and the percentage of nitrogen removal was 98.5 ± 0.2 %. Microbial community analysis indicated that denitrifying methanotrophs dominated (60–70 %) the population of the final sludge. Notably, activity testing and microbial analysis both suggested that heterotrophic denitrifiers existed in the reactor throughout the operation period. After 609 days, the activity testing indicated the nitrogen removal percentage of heterotrophic denitrification was 17 ± 2 % and that of n-damo was 83 ± 2 %. A possible mutualism may be developed between the dominated denitrifying methanotrophs and the associated heterotrophs through cross-feed. Heterotrophs may live on the microbial products excreted by denitrifying methanotrophs and provide growth factors that are required by denitrifying methanotrophs.

Description: Ammonia inhibition of methane fermentation is one of the leading causes of failure of anaerobic digestion reactors. In a batch anaerobic digestion reactor with 429 mM NH 3 -N/L of ammonia, the addition of 25 mM phosphate resulted in an increase in methane production rate. Similar results were obtained with the addition of disodium phosphate in continuous anaerobic digestion using an upflow anaerobic sludge blanket (UASB) reactor. While methane content and production rate decreased in the presence of more than 143 mM NH 3 -N/L of ammonium chloride in UASB, the addition of 5 mM disodium phosphate suppressed ammonia inhibition at 214 mM NH 3 -N/L of ammonium chloride. The addition prevented acetate/propionate accumulation, which might be one of the effects of the phosphate on the ammonia inhibition. The effects on the microbial community in the UASB reactor was also assessed, which was composed of Bacteria involved in hydrolysis, acidogenesis, acetogenesis, and dehydrogenation, as well as Archaea carrying out methanogenesis. The change in the microbial community was observed by ammonia inhibition and the addition of phosphate. The change indicates that the suppression of ammonia inhibition by disodium phosphate addition could stimulate the activity of methanogens, reduce shift in bacterial community, and enhance hydrogen-producing bacteria. The addition of phosphate will be an important treatment for future studies of methane fermentation.

Description: The vitamin B12-dependent riboswitch is a crucial factor that regulates gene transcription to mediate the growth of and vitamin B12 synthesis by Propionibacterium freudenreichii . In this study, the effect of various wavelengths of light on the growth rate and vitamin B12 synthesis was studied. Red, green, and blue light-emitting diodes (LEDs) were selected, and a dark condition was used as the control. The microorganism growth rate was measured using a spectrophotometer and plate counting, while the vitamin B12 content was determined using an HPLC-based method. The optical density at 600 nm (OD 600 ) values indicated that P. freudenreichii grew better under the continuous and discontinuous blue light conditions. Moreover, under the blue light condition, P. freudenreichii tended to have a higher growth rate (0.332 h −1 ) and vitamin B12 synthesis (ca. 10 μg/mL) in tofu wastewater than in dark conditions. HPLC analysis also showed that more methylcobalamin was produced under the blue light conditions than in the other conditions. The cbiB gene transcription results showed that blue light induced the synthesis of this vitamin B12 synthesis enzyme. Moreover, the results of inhibiting the expression of green fluorescent protein indicated that blue light removed the inhibition by the vitamin B12-dependent riboswitch. This method can be used to reduce fermentation time and produce more vitamin B12 in tofu wastewater.

Description: The carotenoid deinoxanthin is a crucial resistance factor against various stresses in the radiation-resistant bacterium Deinococcus radiodurans . Disruption of the gene dr2473 encoding the cytochrome P450 CYP287A1 led to the accumulation of 2-deoxydeinoxanthin in D. radiodurans , demonstrating that CYP287A1 is a novel β-carotene 2-hydroxylase. The dr2473 knockout mutant was shown to be more sensitive to UV radiation and oxidative stress than the wild-type strain D. radiodurans R1, indicating that the C2 alcohol of deinoxanthin is important for antioxidant activity.

Description: Two activated sludge cultures, seeded with activated sludge from the same source, were cultivated for 370 days in synthetic wastewater. Both cultures were transferred weekly to fresh medium; one culture was operated at high dissolved oxygen (DO) (near saturation) and the other at low DO (0.25 mg O 2 /L). There were significant changes in the abundances of bacterial species and phyla present in each culture throughout the 370-day operational period. In the low DO culture, over time, there was a continuously increasing proportion of cells of species known to encode truncated hemoglobins (Hbs). These are the types of Hbs which may enhance delivery of oxygen to the respiratory chain, to enhance ATP production, especially under low aeration conditions. The levels of heme b , the heme found in Vitreoscilla hemoglobin, increased in parallel to the increase in Hb-encoding species, to much higher levels in the low DO culture than in the high DO culture. Specific oxygen uptake rates increased by 3 % for the high DO culture near the end of the 370-day period, while those for the low DO culture increased steadily to a level 28 % higher than that of the starting culture. Thus, imposition of low DO conditions may, due to selection for Hb-expressing species, be useful in developing bacterial communities with enhanced ability to function efficiently in aerobic wastewater treatment, especially under low aeration conditions.

Description: Dewatering of kitchen waste digestate is a key problem to solve so as to increase the application of kitchen waste after anaerobic digestion. In this study, the effects of bioleaching under different hydraulic retention time (HRT = 2, 2.5, and 3 days) on dewaterability of kitchen waste digestate were evaluated. A 12-stage plug flow bioreactor with 180 L working volume was used for digestate bioleaching. The bioleached digestate under different HRTs were collected and dewatered by plate-and-frame filter press. The results showed that the moisture contents of digestate cakes were 67.87 % at 2 days of HRT, 58.06 % at 2.5 days of HRT, and 54.45 % at 3 days of HRT, respectively, indicating the longer the HRT, the lower the moisture content of filter cake. Balanced between the cost and practical need, 2.5 days can be used as the HRT in engineering application. Under the condition of HRT of 2.5 days, the pH, specific resistance to filtration (SRF), capillary suction time (CST), and sedimentation rate of digestate changed from the initial values of 8.08, 210.6 s, 23.4 × 10 12  m kg −1 and 10 % to 3.21, 32.7 s, 2.44 × 10 12  m kg −1 and 76.8 %, respectively. Based on the observations above, the authors conclude that bioleaching technology is an effective method to enhance digestate dewaterability and reduce the cost of subsequent reutilization.

Description: 3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a clinically used anticoagulant drug. Previously, we have developed a method to synthesize PAPS with Escherichia coli crude extracts, which include three overexpressed enzymes and a fourth unidentified protein. The unknown protein degrades adenosine diphosphate ( ADP), the by-product of PAPS synthesis reaction. To further understand and control the process of in vitro enzymatic PAPS synthesis, we decide to identify the fourth protein and develop a defined method to synthesize PAPS using purified enzymes. Here, we show that the purified Nudix hydrolase NudJ degrades ADP at high efficiency and serves as the fourth enzyme in PAPS synthesis. Under the defined condition of PAPS synthesis, all of the 10-mM ADP is hydrolyzed to form adenosine monophosphate (AMP) in a 15-min reaction. ADP is a better substrate for NudJ than adenosine triphosphate (ATP). Most importantly, the purified NudJ does not cleave the product PAPS. The removal of ADP makes the PAPS peak more separable from other components in the chromatographic purification process. This developed enzymatic approach of PAPS production will contribute to the chemoenzymatic synthesis of heparin.

Description: Inactivation of quorum sensing (QS) signal molecules, such as acylhomoserine lactones (AHLs) of pathogenic bacteria, has been proposed as a novel method to combat bacterial diseases in aquaculture. Despite the importance of micro-algae for aquaculture, AHL degradation by bacteria associated with micro-algal cultures has thus far not been investigated. In this study, we isolated Pseudomonas sp. NFMI-T and Bacillus sp. NFMI-C from open cultures of the micro-algae Tetraselmis suecica and Chaetoceros muelleri , respectively. An AHL degradation assay showed that either monocultures or co-cultures of the isolates were able to degrade the AHL N -hexanoyl- l -homoserine lactone. In contrast, only Bacillus sp. NFMI-C was able to inactivate N -hydroxybutanoyl- l -homoserine lactone, the AHL produced by Vibrio campbellii . The isolated bacteria were able to persist for up to 3 weeks in conventionalized micro-algal cultures, indicating that they were able to establish and maintain themselves within open algal cultures. Using gnotobiotic algal cultures, we found that the isolates did not affect growth of the micro-algae from which they were isolated, whereas a mixture of both isolates increased the growth of Tetraselmis and decreased the growth of Chaetoceros . Finally, addition of Bacillus sp. NFMI-C to the rearing water of giant river prawn ( Macrobrachium rosenbergii ) larvae significantly improved survival of the larvae when challenged with pathogenic V. campbellii , whereas it had no effect on larval growth.

Description: Four reactors were initiated to study the effect of inoculum and sulfide type on the simultaneous hydrogen sulfide removal from biogas and nitrogen removal from swine slurry (Ssu-Nir) process. Anaerobic sludge, aerobic sludge, and water were used as inocula, and Na 2 S and biogas were used as a sulfide substrate, respectively. Additionally, 454 pyrosequencing of the 16S rRNA gene was used to explore the bacterial diversity. The results showed that sulfur-oxidizing bacteria ( Thiobacillus , 42.2–84.4 %) were dominant in Ssu-Nir process and led to the excellent performance. Aerobic sludge was more suitable for inoculation of the Ssu-Nir process because it is better for rapidly enriching dominant sulfur-oxidizing bacteria ( Thiobacillus , 54.4 %), denitrifying sulfur-oxidizing bacteria (40.0 %) and denitrifiers (23.9 %). Lower S 2− removal efficiency (72.6 %) and NO 3 − removal efficiency (〈90 %) of the Ssu-Nir process were obtained using biogas as a sulfide substrate than when Na 2 S was used. For the Ssu-Nir process with biogas as the sulfide substrate, limiting H 2 S absorption caused a high relative abundance of sulfur-oxidizing bacteria, Thiobacillus (84.8 %) and Thiobacillus sayanicus (39.6 %), which in turn led to low relative abundance of denitrifiers (1.6 %) and denitrifying sulfur-oxidizing bacteria (24.4 %), low NO 3 − removal efficiency, and eventually poor performance.

Description: Improvement on the bioconversion of cellulosic biomass depends much on the expanded knowledge on the underlying microbial structure and the relevant genetic information. In this study, metagenomic analysis was applied to characterize an enriched mesophilic cellulose-converting consortium, to explore its cellulose-hydrolyzing genes, and to discern genes involved in methanogenesis. Cellulose conversion efficiency of the mesophilic consortium enriched in this study was around 70 %. Apart from methane, acetate was the major fermentation product in the liquid phase, while propionate and butyrate were also detected at relatively high concentrations. With the intention to uncover the biological factors that might shape the varying cellulose conversion efficiency at different temperatures, results of this mesophilic consortium were then compared with that of a previously reported thermophilic cellulose-converting consortium. It was found that the mesophilic consortium harbored a larger pool of putative carbohydrate-active genes, with 813 of them in 54 GH modules and 607 genes in 13 CBM modules. Methanobacteriaceae and Methanosaetaceae were the two methanogen families identified, with a preponderance of the hydrogenotrophic Methanobacteriaceae . In contrast to its relatively high diversity and high abundance of carbohydrate-active genes, the abundance of genes involved in the methane metabolism was comparatively lower in the mesophilic consortium. A biological enhancement on the methanogenic process might serve as an effective option for the improvement of the cellulose bioconversion at mesophilic temperature.

Description: A strain bacterium that is thermophilic, heterotrophic nitrifying, and aerobic denitrifying was isolated and identified as Anoxybacillus contaminans HA for the first time. The identification was based on morphological and physiological characterizations, together with phylogenetic analysis of 16S rDNA sequence. The strain possessed excellent tolerance to high temperatures, with 55 °C as its optimum and 60 °C as viable. Moreover, NH 4 + –N and NO 3 − –N could be efficiently removed under thermophilic and solely aerobic conditions, with little intermediate accumulation. Average removal efficiencies of NH 4 + –N and NO 3 − –N at 55 °C reached 71.0 and 74.7 %, respectively, with removal rates of 5.83 and 32.08 mg l −1  h −1 , respectively. Single-factor experiments suggested that the optimal conditions for both heterotrophic nitrification and aerobic denitrification were glucose as carbon source, NH 4 + –N range of 50–200 mg l −1 , and wide NO 3 − –N range of 200–1000 mg l −1 . These results indicated that strain HA had heterotrophic nitrification and aerobic denitrification abilities, as well as the notable ability to remove ammonium under thermophilic condition. Thus, this strain has potential application in waste-gas treatment.

Description: UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS) are the two enzymes responsible for the biosynthesis of UDP-xylose from UDP-glucose. Several UGDs from bacterial sources, which oxidize UDP-glucose to glucuronic acid, have been found and functionally characterized whereas only few reports on bacterial UXS isoforms exist. Rhodothermus marinus , a halothermophilic bacterium commonly found in hot springs, proved to be a valuable source of carbohydrate active enzymes of biotechnological interest, such as xylanases, mannanases, and epimerases. However, no enzymes of R. marinus involved in the biosynthesis or modification of nucleotide sugars have been reported yet. Herein, we describe the cloning and characterization of two putative UGD (RmUGD1 and RmUGD2) and one UXS (RmUXS) isoform from this organism. All three enzymes could be expressed in recombinant form and purified to near homogeneity. UPLC- and NMR-based activity tests showed that RmUGD1 and RmUXS are indeed active enzymes, whereas no enzymatic activity could be detected by RmUGD2. Both RmUGD1 and RmUXS showed a temperature optimum of 60 °C, with almost no loss of activity after 1 h exposure at 70 °C. No metal ions were required for enzymatic activities. Zn 2+ ions strongly inhibited both enzymes. RmUGD1 showed higher salt tolerance and had a higher pH optimum than RmUXS. Furthermore, RmUGD1 was inhibited by UDP-xylose at higher concentrations. By coupling recombinant RmUXS and RmUGD1, UDP-xylose could be successfully synthesized directly from UDP-glucose. The high activity of the herein described enzymes make RmUGD1 and RmUXS the first thermo-tolerant biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose.

Description: Exploration of cost-effective fermentation substrates for efficient lactate production is an important economic objective. Although some organic nitrogen sources are also cheaper, inorganic nitrogen salts for lactate fermentation have additional advantages in facilitating downstream procedures and significantly improving the commercial competitiveness of lactate production. In this study, we first established an application of diammonium phosphate to replace yeast extract with a reduced 90 % nitrogen cost for a thermotolerant Bacillus coagulans strain. In vivo enzymatic and transcriptional analyses demonstrated that diammonium phosphate stimulates the gene expression of L-lactate dehydrogenase, thus providing higher specific enzyme activity in vivo and increasing L-lactic acid production. This new information provides a foundation for establishing a cost-effective process for polymer-grade L-lactic acid production in an industrial setting.

Description: Ensiling is a feed preservation method of moist forage crops that generally depends on naturally developing lactic acid bacteria to convert water-soluble carbohydrates into organic acids. While bacterial community dynamics have been previously assessed in bench-scale and pilot ensiling facilities, almost no studies have assessed the microbiomes of large-scale silage facilities. This study analyzed bacterial community composition in mature silage from bunker silos in three commercial production centers as related to pH, organic matter, volatile fatty acid composition, and spatial distribution within the ensiling bunker. It revealed significant physicochemical differences between “preserved” regions situated in the center and along the walls of the silage bunkers that were characterized by high concentrations of lactic acid and other volatiles and pH values below 5, and “spoiled” regions in the corners (shoulders) of the bunkers that had low lactic acid concentrations and high pH values. Preserved silage was dominated (〉90 %) by lactic acid bacteria and characterized by high similarity and low taxonomic diversity, whereas spoiled silage had highly diverse microbiomes with low abundances of lactic acid bacteria (〈5 %) that were sometimes characterized by high levels of Enterobacteriaceae . Spatial position had a much stronger impact on the microbial community composition than feedstock type, sampling date, or production center location supporting previous studies demonstrating that ecology and not geography is a major driver of environmental microbiomes.

Description: Microorganisms play a key role in removal of pollutants in constructed wetlands (CWs). The aim of this study was to investigate the composition and diversity of microbes in a full-scale integrated constructed wetland system and examine how microbial assemblages were shaped by the structures and physicochemical properties of the sediments. The microbial assemblages were determined using 16S rRNA high-throughput sequencing. Results showed that the microbial phenotypes were more diverse in the system than in single CWs. The genera of Zoogloea , Comamonas , Thiobacillus , Nitrosospira , Denitratisoma , Azonexus , and Azospira showed relatively high abundances, which contributed to the removal of organic matter and nitrogen. The interactions among the three CWs in series acted a key role in the increase of phylogenetic diversity and high percentage of shared operational taxonomic units. In the system, some core microbes always existed even with the changing environment. Redox potential and NH 4 -N were the important factors affecting the overall microbial community patterns. Total organic carbon had a relatively high impact on some denitrifiers. The results from this study should be useful to better understand the microbial mechanism of wastewater treatment in integrated constructed wetland systems.

Description: Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. Two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N -acetyltransferase (SNAT) catalyzes serotonin to N -acetylserotonin (NAS) followed by the action of N -acetylserotonin O -methyltransferase (ASMT), resulting in the synthesis of O -methylated NAS, also known as melatonin. Attempts to document melatonin production in Escherichia coli have been unsuccessful to date due to either low enzyme activity or inactive ASMT expression. Here, we employed caffeic acid O -methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that has ASMT activity as well. Among several combinations of dual expression cassettes, recombinant E. coli that expressed sheep SNAT with rice COMT produced a high quantity of melatonin, which was measured in a culture medium (1.46 mg/L in response to 1 m m serotonin). This level was several orders of magnitude higher than that produced in transgenic rice and tomato overexpressing sheep SNAT and ASMT , respectively. This heterologous expression system can be widely employed to screen various putative SNAT or ASMT genes from animals and plants as well as to overproduce melatonin in various useful microorganisms.

Description: Persistent use of the diphenyl ether herbicides oxyfluorfen may seriously increase the health risks and ecological safety problems. A newly bacterium R-21 isolated from active soil was able to degrade and utilize oxyfluorfen as the sole carbon source. R-21 was identified as Chryseobacterium aquifrigidense by morphology, physiobiochemical characteristics, and genetic analysis. Under the optimum cultural conditions (pH 6.9, temperature 33.4 °C, and inoculum size 0.2 g L −1 ), R-21 could degrade 92.1 % of oxyfluorfen at 50 mg L −1 within 5 days. During oxyfluorfen degradation, six metabolites were detected and identified by atmospheric pressure gas chromatography coupled to quadrupole–time of flight mass spectrometry and ultra-performance liquid chromatography coupled to quadrupole–time of flight mass spectrometry, and a plausible degradation pathway was deduced. Strain R-21 is a promising potential in bioremediation of oxyfluorfen-contaminated environments.

Description: Fungi comprise organisms like molds, yeasts and mushrooms. They have been used as food or medicine for a long time. A large number of fungal proteins or peptides with diverse biological activities are considered as antibacterial, antifungal, antiviral and anticancer agents. They encompass proteases, ribosome inactivating proteins, defensins, hemolysins, lectins, laccases, ribonucleases, immunomodulatory proteins, and polysaccharopeptides. The target of the present review is to update the status of the various bioactivities of these fungal proteins and peptides and discuss their therapeutic potential.

Description: Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies’ properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography. It is found that acidic charge variant contents were significantly decreased by pH downshift. Then, to reveal the mechanism by which the content of acidic charge variants is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift.

Description: Baeyer-Villiger monooxygenases (BVMOs) are a very well-known and intensively studied class of flavin-dependent enzymes. Their substrate promiscuity, high chemo-, regio-, and enantioselectivity are prerequisites for the use in synthetic chemistry and should pave the way for successful industrial processes. Nonetheless, only a very limited number of industrial relevant transformations are known, mainly due to the lack of BVMOs stability and cofactor dependency. In this review, we focus on novel BVMO-mediated transformations, BVMOs in cascade type reactions, potential industrial applications, and how limitations have been tackled by the community. Special attention will be put on whole-cell immobilization strategies. We emphasize to bridge recent developments in fundamental research to industrial applications.

Description: Lactic acid bacteria (LAB) can interfere with pathogens through different mechanisms; one is the production of biosurfactants, a group of surface-active molecules, which inhibit the growth of potential pathogens. In the present study, biosurfactants produced by Lactobacillus reuteri DSM 17938, Lactobacillus acidophilus DDS-1, Lactobacillus rhamnosus ATCC 53103, and Lactobacillus paracasei B21060 were dialyzed (1 and 6 kDa) and characterized in term of reduction of surface tension and emulsifying activity. Then, aliquots of the different dialyzed biosurfactants were added to Streptococcus mutans ATCC 25175 and Streptococcus oralis ATCC 9811 in the culture medium during the formation of biofilm on titanium surface and the efficacy was determined by agar plate count, biomass analyses, and flow cytometry. Dialyzed biosurfactants showed abilities to reduce surface tension and to emulsifying paraffin oil. Moreover, they significantly inhibited the adhesion and biofilm formation on titanium surface of S . mutans and S . oralis in a dose-dependent way, as demonstrated by the remarkable decrease of cfu/ml values and biomass production. The antimicrobial properties observed for dialyzed biosurfactants produced by the tested lactobacilli opens future prospects for their use against microorganisms responsible of oral diseases.

Description: Addition of MgCl 2 to the culture medium has been found to dramatically increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis . The specific activity of the pullulanase obtained from medium supplemented with MgCl 2 was also higher than that obtained in culture medium without added magnesium ions. In this work, the mechanism of this increase was studied. When cultured in medium without added magnesium ions, B. choshinensis mainly produced a thermolabile, inactive form of pullulanase. The addition of magnesium ions led to the production of a thermostable, active form of pullulanase. Circular dichroism assays revealed considerable differences in secondary structure between the active and inactive pullulanase forms. Transmission electron microscopy suggested that magnesium ion addition inhibits the shedding of cell wall protein (HWP) layers from the cell surface. Quantitative real-time PCR showed that magnesium ion addition represses transcription of HWP . Because the pullulanase gene and HWP have identical promoters, pullulanase gene transcription was also inhibited. These results suggest that when pullulanase is expressed slowly, it tends to fold into an active form.

Description: The heparosan synthase of Escherichia coli K5 is composed of the glycosyltransferases KfiA and KfiC which synthesize the polysaccharide heparosan ( N -acetylheparosan). A third protein, KfiB, is required to stabilize the KfiAC complex in the bacteria and to transport this complex to the inner membrane where the initiation of polymerization occurs. In this report, we fused KfiC with the E. coli trigger factor (TF) to stabilize KfiC, thus activating the enzyme in the absence of KfiB. Different recombinant plasmids were constructed to compare the impact of the presence or absence of KfiB and the presence of the trigger factor as a fusion protein. Several E. coli BL21-derived strains were transformed with recombinant plasmids and cultivated in fed-batch conditions on minimal medium. The bTCA strain overexpressing fused TF-KfiC together with KfiA and KfiD, but lacking KfiB produced 1.5 g/L of total heparosan after 24 h of fed-batch cultivation. This heparosan was essentially intracellular early in the culture, providing evidence that KfiB primarily plays a role in the exportation process. However, over time, heparosan became mostly extracellular, likely due to passive diffusion or partial cell disruption upon product accumulation.

Description: Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans , were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans , in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [ 3 H]phosphatidic acid (PA) released from [ 3 H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.

Description: Tea tree oil (TTO) is a yellow liquid extracted from Melaleuca alternifolia . Although the antimicrobial activity of TTO has been known for a long time, its specific antimicrobial effects and mechanism underlying these remain poorly characterized. The present study investigated the chemical composition of TTO and the dynamics and mechanism of its antimicrobial activities in two bacterial and two fungal strains. Gas chromatography–mass spectrometry analysis identified alkenes and alcohols as the main constituents of TTO. Terpinen-4-ol was the most abundant individual component, accounting for approximately 23 % of the TTO. Poisoned food technique assessment showed that the minimum inhibitory concentrations of TTO for bacterial strains ( Escherichia coli and Staphylococcus aureus ) and fungal strains ( Candida albicans and Aspergillus niger ) were 1.08 and 2.17 mg/mL, respectively. Antimicrobial dynamic curves showed that with increasing concentrations of TTO, the rate of cell killing and the duration of growth lag phase increased correspondingly. These data indicated that TTO produced concentration and time-dependent antimicrobial effects. The minimum bactericidal and fungicidal concentrations of TTO were 2.17, 4.34, and 4.34 against E. coli , S. aureus , and C. albicans , respectively. However, A. niger conidia were not completely eradicated, even after 3 days in the presence of 17.34 mg/mL TTO. Transmission electron microscopy images indicated that TTO penetrated the cell wall and cytoplasmic membrane of all the tested bacterial and fungal strains. TTO may also penetrate fungal organelle membrane. These findings indicated that TTO maybe exerts its antimicrobial effects by compromising the cell membrane, resulting in loss of the cytoplasm and organelle damage, which ultimate leads to cell death.

Description: The gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood of honey bees, a notifiable disease in many countries. Hence, P. larvae can be considered as an entomopathogen of considerable relevance in veterinary medicine. P. larvae is a highly specialized pathogen with only one established host, the honey bee larva. No other natural environment supporting germination and proliferation of P. larvae is known. Over the last decade, tremendous progress in the understanding of P. larvae and its interactions with honey bee larvae at a molecular level has been made. In this review, we will present the recent highlights and developments in P. larvae research and discuss the impact of some of the findings in a broader context to demonstrate what we can learn from studying “exotic” pathogens.

Description: The “reverse methanogenesis” hypothesis as the metabolic pathway of AOM has recently been supported in the novel ANME lineage ANME-2d in denitrifying anaerobic methane oxidation (DAMO). However, no previous studies have experimentally evaluated the reversal of methane oxidation and methane production in this archaea. In the present study, the metabolic reversibility of ANME-2d from AOM to methanogenesis was evaluated using H 2 /CO 2 and acetate as substrates. The results showed that the system produced methane from H 2 /CO 2 but not from acetate. However, the clone library and real-time PCR analysis of the culture showed that both the percentage and quantity of ANME-2d decreased significantly under this condition, while methanogen abundance increased. Further high-throughput sequencing results showed that the archaea community did not change at the fourth day after H 2 /CO 2 was supplied, but changed profoundly after methanogenesis took place for 3 days. The percentage of DAMO archaea in the total archaea decreased obviously, while more methanogens grew up during this period. Comparatively, the bacteria community changed profoundly at the fourth day. These results indicated that ANME-2d might not reverse its metabolism to produce methane from H 2 /CO 2 or acetate. After archaea were returned to DAMO conditions, DAMO activity decreased and the amount of ANME-2d continued to fall, implying that the lineage had suffered from severe injury and required a long recovery time.

Description: In this study, landfill bioreactors were tested to treat the recalcitrant leachate-nitrogen and the impacts of relevant operational parameters on its conversion were comprehensively investigated. We found that the highly diverse microbial community in landfill bioreactors could be substantially affected by increasing biodegradable carbon and oxygen content, which led to the whole system’s intrinsic nitrogen removal capacity increasing from 50 to 70 %, and meanwhile, the contribution of anammox was detected less than 20 %. The sequencing and q-PCR results showed that microbial community in bioreactor was dominated by Proteobacteria (∼35 %) and Acidobacteria (~20 %) during the whole experiment. The abundance of anammox functioning bacteria ( Amx ) kept at a stable level (−2.5 to −2.2 log (copies/16S rRNA)) and was not statistically correlated to the abundance of anammox bacteria. However, significant linear correlation ( p  〈 0.05) was determined between the abundance of nirS and Proteobacteria ; amoA and AOB. Redundancy analysis (RDA) suggested that although oxygen and biodegradable carbon can both impose effects on microbial community structure, only biodegradable carbon content is the determinant in the total nitrogen removal.

Description: Rhodotorula glutinis is capable of synthesizing numerous valuable compounds with a wide industrial usage. Biomass of this yeast constitutes sources of microbiological oils, and the whole pool of fatty acids is dominated by oleic, linoleic, and palmitic acid. Due to its composition, the lipids may be useful as a source for the production of the so-called third-generation biodiesel. These yeasts are also capable of synthesizing carotenoids such as β-carotene, torulene, and torularhodin. Due to their health-promoting characteristics, carotenoids are commonly used in the cosmetic, pharmaceutical, and food industries. They are also used as additives in fodders for livestock, fish, and crustaceans. A significant characteristic of R. glutinis is its capability to produce numerous enzymes, in particular, phenylalanine ammonia lyase (PAL). This enzyme is used in the food industry in the production of l -phenylalanine that constitutes the substrate for the synthesis of aspartame—a sweetener commonly used in the food industry.

Description: Management of manure containing veterinary antibiotics is a major concern in anaerobic treatment systems because of their possible adverse effects on microbial communities. Therefore, the aim of study was to investigate how oxytetracycline (OTC) influences bacteria and acetoclastic and hydrogenotrophic methanogens under varying operational conditions in OTC-medicated and non-medicated anaerobic cow manure digesters. Concentrations of OTC and its metabolites throughout the anaerobic digestion were determined using ultraviolet-high-performance liquid chromatography (UV-HPLC) and tandem liquid chromatography-mass spectrometry (LC/MS/MS), respectively. Fluorescent in situ hybridization, denaturing gradient gel electrophoresis, cloning, and sequencing analyses were used to monitor changes in microbial community structures. According to the results of analytical and molecular approaches, operating conditions highly influence active microbial community dynamics and associate with biogas production and elimination of OTC and its metabolites during anaerobic digestion of cow manure in the presence of an average initial concentration of 2.2 mg OTC/L. The impact of operating conditions has a drastic effect on acetoclastic methanogens than hydrogenotrophic methanogens and bacteria.

Description: Thermophilic Bacillus coagulans WCP10-4 is found to be able to convert cellobiose to optically pure l -lactic acid. Its β-glucosidase activity is detected in whole cells (7.3 U/g dry cells) but not in culture medium, indicating the intracellular location of the enzyme. Its β-glucosidase activity is observed only when cultured using cellobiose as the sole carbon source, indicating that the expression of this enzyme is tightly regulated in cells. The enzyme is most active at 50 °C and pH 7.0. The supplement of external β-glucosidase during fermentation of cellobiose (106 g/l) by B. coagulans WCP10-4 increased the fermentation time from 21 to 23 h and decreased the lactic acid yield from 96.1 to 92.9 % compared to the control without β-glucosidase supplementation. B. coagulans WCP10-4 converted 200 g/l of cellobiose to 196.3 g/l of l -lactic acid at a yield of 97.8 % and a productivity of 7.01 g/l/h. This result shows that B. coagulans WCP10-4 is a highly efficient strain for converting cellobiose to l -lactic acid without the need of supplementing external β-glucosidases.

Description: Pseudomonas aeruginosa and Staphylococcus aureus are versatile bacterial pathogens and common etiological agents in polymicrobial infections. Microbial communities containing both of these pathogens are shaped by interactions ranging from parasitic to mutualistic, with the net impact of these interactions in many cases resulting in enhanced virulence. Polymicrobial communities of these organisms are further defined by multiple aspects of the host environment, with important implications for disease progression and therapeutic outcomes. This mini-review highlights the impact of these interactions on the host and individual pathogens, the molecular mechanisms that underlie these interactions, and host-specific factors that drive interactions between these two important pathogens.

Description: The extrusion technology of blends formed by compounds with different physicochemical properties often results in new materials that present properties distinctive from its original individual constituents. Here, we report the use of melt extrusion of blends made from low-cost materials to produce a biodegradable foam suitable for use as an inoculant carrier of plant growth-promoting bacteria (PGPB). Six formulations were prepared with variable proportions of the raw materials; the resulting physicochemical and structural properties are described, as well as formulation performance in the maintenance of bacterial viability during 120 days of storage. Differences in blend composition influenced foam density, porosity, expansion index, and water absorption. Additionally, differences in the capability of sustaining bacterial viability for long periods of time were more related to the foam composition than to the resulting physicochemical characteristics. Microscopic analyses showed that the inoculant bacteria had firmly attached to the extruded material by forming biofilms. Inoculation assays using maize plants demonstrated that the bacteria attached to the extruded foams could survive in the soil for up to 10 days before maize sowing, without diminishing its ability to promote plant growth. The results presented demonstrate the viability of the new matrix as a biotechnological material for bacterial delivery not only in agriculture but also in other biotechnological applications, according to the selected bacterial strains.

Description: The bacterial dioxygenation of mono- or polycyclic aromatic compounds is an intensely studied field. However, only in a few cases has the repeated dioxygenation of a substrate possessing more than a single aromatic ring been described. We previously characterized the aryl-hydroxylating dioxygenase BphA-B4h, an artificial hybrid of the dioxygenases of the biphenyl degraders Burkholderia xenovorans LB400 and Pseudomonas sp. strain B4-Magdeburg, which contains the active site of the latter enzyme, as an exceptionally powerful biocatalyst. We now show that this dioxygenase possesses a remarkable capacity for the double dioxygenation of various bicyclic aromatic compounds, provided that they are carbocyclic. Two groups of biphenyl analogues were examined: series A compounds containing one heterocyclic aromatic ring and series B compounds containing two homocyclic aromatic rings. Whereas all of the seven partially heterocyclic biphenyl analogues were solely dioxygenated in the homocyclic ring, four of the six carbocyclic bis-aryls were converted into ortho , meta -hydroxylated bis-dihydrodiols. Potential reasons for failure of heterocyclic dioxygenations are discussed. The obtained bis-dihydrodiols may, as we also show here, be enzymatically re-aromatized to yield the corresponding tetraphenols. This opens a way to a range of new polyphenolic products, a class of compounds known to exert multiple biological activities. Several of the obtained compounds are novel molecules.

Description: Biofertilizers can help improve soil quality, promote crop growth, and sustain soil health. The photosynthetic bacterium Rhodopseudomonas palustris strain PS3 (hereafter, PS3), which was isolated from Taiwanese paddy soil, can not only exert beneficial effects on plant growth but also enhance the efficiency of nutrient uptake from applied fertilizer. To produce this elite microbial isolate for practical use, product development and formulation are needed to permit the maintenance of the high quality of the inoculant during storage. The aim of this study was to select a suitable formulation that improves the survival and maintains the beneficial effects of the PS3 inoculant. Six additives (alginate, polyethylene glycol [PEG], polyvinylpyrrolidone-40 [PVP], glycerol, glucose, and horticultural oil) were used in liquid-based formulations, and their capacities for maintaining PS3 cell viability during storage in low, medium, and high temperature ranges were evaluated. Horticultural oil (0.5 %) was chosen as a potential additive because it could maintain a relatively high population and conferred greater microbial vitality under various storage conditions. Furthermore, the growth-promoting effects exerted on Chinese cabbage by the formulated inoculants were significantly greater than those of the unformulated treatments. The fresh and dry weights of the shoots were significantly increased, by 10–27 and 22–40 %, respectively. Horticultural oil is considered a safe, low-cost, and easy-to-process material, and this formulation would facilitate the practical use of strain PS3 in agriculture.

Description: In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus , creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency ( k cat / K m (app) ) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m (app) . This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

Description: Polyhydroxyalkanoates (PHAs) are bio-based, biodegradable polyesters that can be produced from organic-rich waste streams using mixed microbial cultures (MMCs). To maximize PHA production, MMCs are enriched for bacteria with a high polymer storage capacity through the application of aerobic dynamic feeding (ADF) in a sequencing batch reactor (SBR), which consequently induces a feast-famine metabolic response. Though the feast-famine response is generally understood empirically at a macro-level, the molecular level is less refined. The objective of this study was to investigate the microbial community composition and proteome profile of an enriched MMC cultivated on fermented dairy manure. The enriched MMC exhibited a feast-famine response and was capable of producing up to 40 % (wt. basis) PHA in a fed-batch reactor. High-throughput 16S rRNA gene sequencing revealed a microbial community dominated by Meganema , a known PHA-producing genus not often observed in high abundance in enrichment SBRs. The application of the proteomic methods two-dimensional electrophoresis and LC-MS/MS revealed PHA synthesis, energy generation, and protein synthesis prominently occurring during the feast phase, corroborating bulk solution variable observations and theoretical expectations. During the famine phase, nutrient transport, acyl-CoA metabolism, additional energy generation, and housekeeping functions were more pronounced, informing previously under-determined MMC functionality under famine conditions. During fed-batch PHA production, acetyl-CoA acetyltransferase and PHA granule-bound phasin proteins were in increased abundance relative to the SBR, supporting the higher PHA content observed. Collectively, the results provide unique microbial community structural and functional insight into feast-famine PHA production from waste feedstocks using MMCs.