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Applied Microbiology and Biotechnology (v.95, #3)
Microbial communities in large-scale wood piles and their effects on wood quality and the environment by Matthias Noll; Raida Jirjis (pp. 551-563).
The demand of renewable energy sources, i.e. biomass, is steadily increasing worldwide to reduce the need of fossil energy sources. Biomass such as energy crops, woody species, forestry and agricultural residues are the most common renewable energy sources. Due to uneven demand for wood fuel, the material is mostly stored outdoors in chip piles or as logs until utilisation. Storage of biomass is accompanied by chemical, physical and biological processes which can significantly reduce the fuel quality. However, heating plants require high-quality biomass to ensure efficient operation, thereby minimising maintenance costs. Therefore, optimised storage conditions and duration times for chipped wood and tree logs have to be found. This paper aims at reviewing available knowledge on the pathways of microbial effects on stored woody biomass and on investigations of the fungal and bacterial community structure and identity. Moreover, potential functions of microorganisms present in wood chip piles and logs are discussed in terms of (1) reduction of fuel quality, (2) catalysing self-ignition processes, and (3) constituting health risk and unfriendly work environment.
Keywords: Bacteria; Fuel quality; Fungi; Self-ignition; Storage; Wood piles
Pectin-rich biomass as feedstock for fuel ethanol production by Meredith C. Edwards; Joy Doran-Peterson (pp. 565-575).
The USA has proposed that 30 % of liquid transportation fuel be produced from renewable resources by 2030 (Perlack and Stokes 2011). It will be impossible to reach this goal using corn kernel-based ethanol alone. Pectin-rich biomass, an under-utilized waste product of the sugar and juice industry, can augment US ethanol supplies by capitalizing on this already established feedstock. Currently, pectin-rich biomass is sold (at low value) as animal feed. This review focuses on the three most studied types of pectin-rich biomass: sugar beet pulp, citrus waste and apple pomace. Fermentations of these materials have been conducted with a variety of ethanologens, including yeasts and bacteria. Escherichia coli can ferment a wide range of sugars including galacturonic acid, the primary component of pectin. However, the mixed acid metabolism of E. coli can produce unwanted side products. Saccharomyces cerevisiae cannot naturally ferment galacturonic acid nor pentose sugars but has a homoethanol pathway. Erwinia chrysanthemi is capable of degrading many of the cell wall components of pectin-rich materials, including pectin. Klebsiella oxytoca can metabolize a diverse array of sugars including cellobiose, one degradation product of cellulose. However, both E. chrysanthemi and K. oxytoca produce side products during fermentation, similar to E. coli. Using pectin-rich residues from industrial processes is beneficial because the material is already collected and partially pretreated to facilitate enzymatic deconstruction of the plant cell walls. Using biomass already produced for other purposes is an attractive practice because fewer greenhouse gases (GHG) will be anticipated from land-use changes.
Keywords: Pectin; Ethanol; Biofuels; Fermentation
Recent developments in yeast cell surface display toward extended applications in biotechnology by Tsutomu Tanaka; Ryosuke Yamada; Chiaki Ogino; Akihiko Kondo (pp. 577-591).
Yeasts are promising hosts for industrial bio-refinery applications. In yeast cell surface displays, functional proteins, such as cellulases or lipases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae is the most commonly used yeast for cell surface display. Engineered yeasts have been utilized for a variety of applications, such as bioethanol production, chemicals synthesis, adsorption of environmental pollutants, and protein evolution. Here, we summarize recent developments in yeast cell surface display techniques for bio-refinery applications, including methods using hosts such as Pichia pastoris, Yarrowia lipolytica, and S. cerevisiae, focusing on the characteristics of anchor proteins and applications.
Keywords: Cell surface display; Yeast; Anchor; Cell surface engineering
Structure, biosynthesis, and properties of kurstakins, nonribosomal lipopeptides from Bacillus spp. by Max Béchet; Thibault Caradec; Walaa Hussein; Ahmed Abderrahmani; Marlène Chollet; Valérie Leclère; Thomas Dubois; Didier Lereclus; Maude Pupin; Philippe Jacques (pp. 593-600).
A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. Kurstakins are lipoheptapeptides displaying antifungal activities against Stachybotrys charatum. Recently, the biosynthesis mechanism, the regulation of this biosynthesis and the potential new properties of kurstakins were described in the literature. In addition, kurstakins were also detected in other species belonging to Bacillus genus such as Bacillus cereus. This mini-review gathers all the information about these promising bioactive molecules.
Keywords: Kurstakins; Lipopeptides; Bacillus cereus ; Bacillus thuringiensis ; NRPS; Spreading
Surviving in the presence of sulphur dioxide: strategies developed by wine yeasts by Benoit Divol; Maret du Toit; Edward Duckitt (pp. 601-613).
Sulphur dioxide has been used as a common preservative in wine since at least the nineteenth century. Its use has even become essential to the making of quality wines because of its antioxidant, antioxidasic and antiseptic properties. The chemistry of SO2 in wine is fairly complex due to its dissociation into different species and its binding to other compounds produced by yeasts and bacteria during fermentation. The only antiseptic species is the minute part remaining as molecular SO2. The latter concentration is both dependent on pH and concentration of free bisulphite. However, certain yeast species have developed cellular and molecular mechanisms as a response to SO2 exposure. Some of these mechanisms are fairly complex and have only been investigated recently, at least for the molecular mechanisms. They include sulphite reduction, sulphite oxidation, acetaldehyde production, sulphite efflux and the entry into viable but not culturable state, as the ultimate response. In this review, the chemistry of SO2 in wine is explained together with the impact of SO2 on yeast cells. The different defence mechanisms are described and discussed, mostly based on current knowledge available for Saccharomyces cerevisiae.
Keywords: Yeast; Wine; Sulphur dioxide; Cellular and molecular response; VBNC state
Synthesis of highly water-soluble feruloyl diglycerols by esterification of an Aspergillus niger feruloyl esterase by Masaki Kikugawa; Moriyasu Tsuchiyama; Kenji Kai; Tatsuji Sakamoto (pp. 615-622).
Ferulic acid (FA) is a component of plant cell walls that has applications in food, cosmetic, and health products, but its applications are limited by its high insolubility. We synthesized water-soluble FA derivatives by esterification of FA with diglycerol (DG) using feruloyl esterase purified from a commercial enzyme preparation produced by Aspergillus niger. The major reaction product, FA-DG1, was determined to be γ-feruloyl-α,α′-DG by NMR and electrospray ionization mass spectrometry analyses. FA-DG1 is a sticky liquid whose water solubility (>980 mg/ml) is dramatically higher than that of FA (0.69 mg/ml). Suitable conditions for esterification of FA with DG were 100 mg of FA in the presence of 1 g of DG and 0.1 ml of 1 M phosphate buffer (pH 6.0) at 50 °C under reduced pressure. Under these conditions, 168 mg of feruloyl DGs (FA-DG1, 2, and 3) was obtained, corresponding to a 95 % conversion rate of FA. We also developed a batch method which resulted in synthesis of 729 mg of feruloyl DGs and 168 mg of diferuloyl DGs from 600 mg of FA and 1 g of DG (corresponding to conversion of 69 % of the FA to feruloyl DGs and 21 % of the FA to diferuloyl DGs). As an anti-oxidant, feruloyl DGs were essentially equal to FA and butyl hydroxytoluene in scavenging 1,1-diphenyl-2-picrylhydrazyl radicals. In contrast, the scavenging abilities of diferuloyl DGs were twice those of feruloyl DGs.
Keywords: Ferulic acid; Feruloyl esterase; Esterification; Feruloyl diglycerol; Fed-batch reaction
Development of a bioprocess to convert PET derived terephthalic acid and biodiesel derived glycerol to medium chain length polyhydroxyalkanoate by Shane T. Kenny; Jasmina Nikodinovic Runic; Walter Kaminsky; Trevor Woods; Ramesh P. Babu; Kevin E. O’Connor (pp. 623-633).
Sodium terephthalate (TA) produced from a PET pyrolysis product and waste glycerol (WG) from biodiesel manufacture were supplied to Pseudomonas putida GO16 in a fed-batch bioreactor. Six feeding strategies were employed by altering the sequence of TA and WG feeding. P. putida GO16 reached 8.70 g/l cell dry weight (CDW) and 2.61 g/l PHA in 48 h when grown on TA alone. When TA and WG were supplied in combination, biomass productivity (g/l/h) was increased between 1.3- and 1.7-fold and PHA productivity (g/l/h) was increased 1.8- to 2.2-fold compared to TA supplied alone. The monomer composition of the PHA accumulated from TA or WG was predominantly composed of 3-hydroxydecanoic acid. PHA monomers 3-hydroxytetradeeanoic acid and 3-hydroxytetradecenoic acid were not present in PHA accumulated from TA alone but were present when WG was supplied to the fermentation. When WG was either the sole carbon source or the predominant carbon source supplied to the fermentation the molecular weight of PHA accumulated was lower compared to PHA accumulated when TA was supplied as the sole substrate. Despite similarities in data for the properties of the polymers, PHAs produced with WG present in the PHA accumulation phase were tacky while PHA produced where TA was the sole carbon substrate in the polymer accumulation phase exhibited little or no tackiness at room temperature. The co-feeding of WG to fermentations allows for increased utilisation of TA. The order of feeding of WG and TA has an effect on TA utilisation and polymer properties.
Keywords: Waste; Glycerol; Polyhydroxyalkanoate; Terephthalic acid; Polyethylene terephthalate
Asymmetric bioreduction of activated alkenes by a novel isolate of Achromobacter species producing enoate reductase by Yan-Jie Liu; Xiao-Qiong Pei; Hui Lin; Ping Gai; Yu-Chang Liu; Zhong-Liu Wu (pp. 635-645).
The strain Achromobacter sp. JA81, which produced enoate reductase, was applied in the asymmetric reduction of activated alkenes. The strain could catalyze the bioreduction of alkenes to form enantiopure (R)-β-aryl-β-cyano-propanoic acids, a precursor of (R)-γ-amino butyric acids, including the pharmaceutically active enantiomer of the chiral drug (R)-baclofen with excellent enantioselectivity. It could catalyze as well the stereoselective bioreduction of other activated alkenes such as cyclic imides, β-nitro acrylates, and nitro-alkenes with up to >99 % ee and >99 % conversion. The draft genome sequencing of JA81 revealed six putative old yellow enzyme homologies, and the transcription of one of them, Achr-OYE3, was detected using reverse transcription polymerase chain reaction. The recombinant Escherichia coli expressing Achr-OYE3 displayed enoate reductase activity toward (Z)-3-cyano-3-phenyl-propenoic acid (2a).
Keywords: Asymmetric reduction; Old yellow enzyme; Enoate reductase; Activated alkene; Achromobacter sp.; Baclofen
Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain by Caroline Wilde; Nicholas D. Gold; Nancy Bawa; José Humberto M. Tambor; Lina Mougharbel; Reginald Storms; Vincent J. J. Martin (pp. 647-659).
Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.
Keywords: Saccharomyces cerevisiae ; β-Glucosidase; Ethanol; Cell-surface display
Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop by Ya-Shan Cheng; Tzu-Ping Ko; Jian-Wen Huang; Tzu-Hui Wu; Cheng-Yen Lin; Wenhua Luo; Qian Li; Yanhe Ma; Chun-Hsiang Huang; Andrew H.-J. Wang; Je-Ruei Liu; Rey-Ting Guo (pp. 661-669).
Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable β-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The k cat and K m values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G–substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application.
Keywords: Thermostability; Endoglucanase; Crystal structure; Protein engineering; Enzyme kinetics
Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris by Byung-Kwon Choi; Shannon Warburton; Heping Lin; Rohan Patel; Istvan Boldogh; Meehl Meehl; Marc d’Anjou; Liza Pon; Terrance A. Stadheim; Natarajan Sethuraman (pp. 671-682).
Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.
Keywords: Oligosaccharyltransferase; L. major STT3D; N-glycan site occupancy; Pichia pastoris
Overproduction and easy recovery of target gene products from cyanobacteria, photosynthesizing microorganisms by Munehiko Asayama (pp. 683-695).
New cyanobacterial expression vectors, possessing an origin of replication that functions in a broad range of Gram-negative bacteria, were constructed. To inspect the shuttle vectors, the gene gfp was cloned downstream from the expression control element (ECE) originating from the regulatory region of the Microcystis aeruginosa gene psbA2 (for photosystem II D1 protein), and the vectors were introduced into three kinds of cyanobacteria (Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, and Limnothrix/Pseudanabaena sp. ABRG5-3) by conjugation. Multiple copy numbers of the expression vectors (in the range of 14–25 copies per cell) and a high expression of green fluorescent protein (GFP) at the RNA/protein level were observed in the cyanobacterial transconjugants. Importantly, GFP was observed in a supernatant from the autolysed transconjugants of ABRG5-3 and easily collected from the supernatant without centrifugation and/or further cell lysis. These results indicate the vectors together with the recombinant cells to be useful for overproducing and recovering target gene products from cyanobacteria.
Keywords: AU-box sequence; Auto cell lysis cyanobacteria; Conjugation; Expression vector; Light-responsive psbA promoter
Detection of an internal translation activity in the 5′ region of Bombyx mori infectious flacherie virus by Mingqian Li; Nana Man; Haihong Qiu; Shunfeng Cai; Xinyi He; Xiangkang He; Xingmeng Lu (pp. 697-705).
The 5′ untranslated region plays an important role in positive-sense single-stranded RNA virus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation and is applied to simultaneously express several proteins. Infectious flacherie virus (IFV) is a positive-sense single-stranded RNA virus; however, the IRES function is still not proved. To investigate whether the sequences of IFV contain IRES activity, a series of bicistronic reporter (DsRed and enhanced green fluorescent protein) recombinant baculoviruses were constructed to infect the insect cells and silkworm using the Bombyx mori baculovirus expression system. Results showed that the upstream 311, 323, 383, 551, and 599 nt have IRES activity except for the 155-nt region in BmN cells. More importantly, the tetraloop structure containing region between 551 and 599 nt appeared to be responsible for the enhanced IRES activity in different insect cell lines and silkworm. These results indicated that the IRES activity is not species specific and tissue specific. Therefore, our findings may provide the basis for the simultaneous expression of two or various different genes under the same promoter in baculovirus expression system.
Keywords: Bombyx mori ; Infectious flacherie virus; Internal ribosome entry site; Baculovirus; Expression system
Expression analysis of the spi gene in the pock-forming plasmid pSA1.1 from Streptomyces azureus and localization of its product during differentiation by Katusmi Doi; Yukiko Ohyama; Eiji Yokoyama; Takashi Nishiyama; Yasuhiro Fujino; Yuko Nagayoshi; Toshihisa Ohshima; Seiya Ogata (pp. 707-716).
The sporulation inhibitory gene spi in the pock-forming conjugative plasmid pSA1.1 of Streptomyces azureus was introduced into cells via a high or low copy number vector to examine the effect of gene dosage on the growth of Streptomyces lividans TK24 as a host. In transformants carrying a high spi copy number, nutrient mycelial growth was inhibited, as was morphological differentiation from substrate mycelium to aerial mycelium on solid media. The degree of inhibition depended on the spi gene dosage, but the presence of pSA1.1 imp genes, which encode negative repressor proteins for spi, relieved the inhibition. Confocal images of Spi tagged with enhanced green fluorescent protein in cells on solid media revealed that spi expression was initiated at the time of elongation of substrate mycelium, that its expression increased dramatically at septation in aerial hyphae, and that the expression was maximal during prespore formation. Expression of spi covered the whole of the hyphae, and the level of expression at the tip of the hyphae during prespore formation was about sixfold greater than during substrate mycelial growth and threefold greater than during aerial mycelial growth. Thus, localized expression of spi at particular times may inhibit sporulation until triggering imp expression to repress its inhibitory effects.
Keywords: Plasmid; Streptomyces ; Conjugation; Pock formation; DNA translocation; Morphological differentiation
Differential proteomic analysis of the response of Stenotrophomonas maltophilia to imipenem by Laurence Van Oudenhove; Kris De Vriendt; Jozef Van Beeumen; Paola Sandra Mercuri; Bart Devreese (pp. 717-733).
This study represents two different large-scale proteomic experiments analyzing the antibiotic response and the mechanisms of production of β-lactamases in the nosocomial pathogen Stenotrophomonas maltophilia. Two-dimensional gel electrophoresis on the cytoplasmic protein fraction, together with iTRAQ® differential labeling and 2-D liquid chromatographic separation (2D-LC) MS/MS on the enriched membrane protein fraction, revealed 73 proteins with a change in abundance upon imipenem challenge. These proteins belong to several different functional pathways. We observe an increase in β-lactamase production as well as in proteins important for their function in the periplasm. The up-regulation of the L1 and L2 β-lactamases, along with their activator LysR transcriptional factor AmpR, is linked to an increase in proteins responsible for peptidoglycan remodeling and stress response. The interesting identification of an increase in abundance after treatment of the two-component GGDEF signaling protein and an integral membrane sensor signal transduction histidine kinase, indicates that induction of the β-lactamases is not restricted to the ampR-ampD-ampG pathway. This is the first proteomic study in S. maltophilia upon imipenem stimulation to further unravel the cellular adaptation resulting in β-lactamase production.
Keywords: Antibiotics; Resistance; Opportunistic pathogen; 2-DE; LCMS
Impact of each individual component of the mutated PTSNag on glucose uptake and phosphorylation in Ralstonia eutropha G+1 by Matthias Raberg; Chlud Kaddor; Bernhard Kusian; Gertrud Stahlhut; Radka Budinova; Nikola Kolev; Botho Bowien; Alexander Steinbüchel (pp. 735-744).
A recent study of the UV-generated glucose-utilizing mutant Ralstonia eutropha G+1 comprising transcriptomic and proteomic analyses revealed clear evidence that glucose is transported by the N-acetylglucosamine-specific phosphotransferase system (PTSNag), which is overexpressed in this mutant due to a derepression of the encoding nag operon by an identified insertion mutation in nagR (Raberg et al., Appl Environ Microbiol 77:2058–2070, 2011). The inability of the defined deletion mutant R. eutropha G+1∆nagFEC to utilize glucose confirms this finding. Furthermore, a missense mutation in nagE (membrane component comprising the cell membrane spanning EIICNag and the cytosolic domain EIIBNag) was identified, which yields a substitution of an alanine by threonine at aa 153 of NagE and may affect glucose specificity of the mutated PTSNag in R. eutropha G+1. The investigation of various generated deletion and substitution mutants of R. eutropha H16 and G+1 in this study was able to elucidate these phenomena. It could be shown that the porin NagC, encoded by nagC being part of the nag operon, is not necessary, while NagE is required and is probably responsible for glucose transport through the cell membrane. The intracellular phosphorylation of glucose is obviously mediated by the glucokinase GLK and not by NagF (cytosolic component comprising the three soluble domains EIIANag, HPrNag, and EINag). Our data clearly indicate that the derepression of the nag operon is essential for glucose uptake. The point mutation in NagE is not an essential prerequisite for glucose transport although it increased glucose transport as observed in this study.
Keywords: Ralstonia eutropha ; Glucose utilization; PHB; Carbohydrate phosphotransferase system (PTS); nag operon
Effect of water activity and protective solutes on growth and subsequent survival to air-drying of Lactobacillus and Bifidobacterium cultures by Claude P. Champagne; Yves Raymond; Jean-Paul Simon (pp. 745-756).
Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities (a w) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a w = 0.96 or 0.99 were then recovered by centrifugation, added to a sucrose–phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a w = 0.96. Growing the cells in complex media adjusted at a w = 0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose–phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures.
Keywords: Sucrose; Betaine; Osmotic stress; Lactobacilli; Bifidobacteria; Water activity
The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a by Abigail A. Adebusuyi; Angela Y. Smith; Murray R. Gray; Julia M. Foght (pp. 757-766).
Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of 14C-phenanthrene to radiolabeled polar metabolites and 14CO2. The emhB − WEN strain produced approximately twice as much 14CO2 and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of 14C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.
Keywords: RND efflux pump; Phenanthrene biodegradation; Environmental PAH pollutants; Pseudomonas fluorescens LP6a; Dibenzothiophene
Step-by-step strategy for protein enrichment and proteome characterisation of extracellular polymeric substances in wastewater treatment systems by Ana F. Silva; Gilda Carvalho; Renata Soares; Ana V. Coelho; M. Teresa Barreto Crespo (pp. 767-776).
Extracellular polymeric substances (EPS) are keys in biomass aggregation and settleability in wastewater treatment systems. In membrane bioreactors (MBR), EPS are an important factor as they are considered to be largely responsible for membrane fouling. Proteins were shown to be the major component of EPS produced by activated sludge and to be correlated with the properties of the sludge, like settling, hydrophobicity and cell aggregation. Previous EPS proteomic studies of activated sludge revealed several problems, like the interference of other EPS molecules in protein analysis. In this study, a successful strategy was outlined to identify the proteins from soluble and bound EPS extracted from activated sludge of a lab-scale MBR. EPS samples were first subjected to pre-concentration through lyophilisation, centrifugal ultrafiltration or concentration with a dialysis membrane coated by a highly absorbent powder of polyacrylate-polyalcohol, preceded or not by a dialysis step. The highest protein concentration factors were achieved with the highly absorbent powder method without previous dialysis step. Four protein precipitation methods were then tested: acetone, trichloroacetic acid (TCA), perchloric acid and a commercial kit. Protein profiles were compared in 4–12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. Both acetone and TCA should be applied for the highest coverage for soluble EPS proteins, whereas TCA was the best method for bound EPS proteins. All visible bands of selected profiles were subjected to mass spectrometry analysis. A high number of proteins (25–32 for soluble EPS and 17 for bound EPS) were identified. As a conclusion of this study, a workflow is proposed for the successful proteome characterisation of soluble and bound EPS from activated sludge samples.
Keywords: Extracellular polymeric substances (EPS); Extracellular proteins; Wastewater treatment plants; Activated sludge; SDS-PAGE; Protein identification
A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU) by Sayaka Ohno; Hironori Okano; Yasunori Tanji; Akiyoshi Ohashi; Kazuya Watanabe; Ken Takai; Hiroyuki Imachi (pp. 777-788).
The evaluation of bacteriophage (phage) host range is a significant issue in understanding phage and prokaryotic community interactions. However, in conventional methods, such as plaque assay, target host strains must be isolated, although almost all environmental prokaryotes are recalcitrant to cultivation. Here, we introduce a novel phage host range evaluation method using fluorescently labeled phages (the FLP method), which consists of the following four steps: (i) Fluorescently labeled phages are added to a microbial consortium, and host cells are infected and fluorescently labeled. (ii) Fluorescent cells are sorted by fluorescence-activated cell sorting. (iii) 16S rRNA gene sequences retrieved from sorted cells are analyzed, and specific oligonucleotide probes for fluorescence in situ hybridization (FISH) are designed. (iv) Cells labeled with both fluorescently labeled phage and FISH probe are identified as host cells. To verify the feasibility of this method, we used T4 phage and Escherichia coli as a model. We first used nucleic acid stain reagents for phage labeling; however, the reagents also stained non-host cells. Next, we employed the Click-iT EdU (5-ethynyl-2′-deoxyuridine) assay kit from Invitrogen for phage labeling. Using EdU-labeled T4 phage, we could specifically detect E. coli cells in a complex microbial consortium from municipal sewage. We also confirmed that FISH could be applied to the infected E. coli cells. These results suggest that this FLP method using the EdU assay kit is a useful method for evaluating phage host range and may have a potential application for various types of phages, even if their prokaryotic hosts are currently unculturable.
Keywords: Phage; Host range; EdU; Click chemistry; Fluorescence in situ hybridization (FISH)
Biodegradation of polycyclic aromatic hydrocarbons by a halophilic microbial consortium by Seyed Mohammad Mehdi Dastgheib; Mohammad Ali Amoozegar; Khosro Khajeh; Mahmoud Shavandi; Antonio Ventosa (pp. 789-798).
In this study we investigated the phenanthrene degradation by a halophilic consortium obtained from a saline soil sample. This consortium, named Qphe, could efficiently utilize phenanthrene in a wide range of NaCl concentrations, from 1% to 17% (w/v). Since none of the purified isolates could degrade phenanthrene, serial dilutions were performed and resulted in a simple polycyclic aromatic hydrocarbon (PAH)-degrading culture named Qphe-SubIV which was shown to contain one culturable Halomonas strain and one unculturable strain belonging to the genus Marinobacter. Qphe-SubIV was shown to grow on phenanthrene at salinities as high as 15% NaCl (w/v) and similarly to Qphe, at the optimal NaCl concentration of 5% (w/v), could degrade more than 90% of the amended phenanthrene in 6 days. The comparison of the substrate range of the two consortiums showed that the simplified culture had lost the ability to degrade chrysene but still could grow on other polyaromatic substrates utilized by Qphe. Metabolite analysis by HPLC and GC–MS showed that 2-hydroxy 1-naphthoic acid and 2-naphthol were among the major metabolites accumulated in the Qphe-SubIV culture media, indicating that an initial dioxygenation step might proceed at C1 and C2 positions. By investigating the growth ability on various substrates along with the detection of catechol dioxygenase gene, it was postulated that the uncultured Marinobacter strain had the central role in phenanthrene degradation and the Halomonas strain played an auxiliary role in the culture by utilizing phenanthrene metabolites whose accumulation in the media could be toxic.
Keywords: Polycyclic aromatic hydrocarbons (PAHs); Phenanthrene; Biodegradation; Halophile; Marinobacter ; Halomonas ; Consortium
Influence of the sulfur species reactivity on biofilm conformation during pyrite colonization by Acidithiobacillus thiooxidans by René H. Lara; J. Viridiana García-Meza; Roel Cruz; Donato Valdez-Pérez; Ignacio González (pp. 799-809).
Massive pyrite (FeS2) electrodes were potentiostatically modified by means of variable oxidation pulse to induce formation of diverse surface sulfur species (S n 2−, S0). The evolution of reactivity of the resulting surfaces considers transition from passive (e.g., Fe1−x S2) to active sulfur species (e.g., Fe1−x S2−y , S0). Selected modified pyrite surfaces were incubated with cells of sulfur-oxidizing Acidithiobacillus thiooxidans for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the attached cells density and their exopolysaccharides were analyzed by confocal laser scanning microscopy (CLMS) and atomic force microscopy (AFM) on bio-oxidized surfaces; additionally, S n 2−/S0 speciation was carried out on bio-oxidized and abiotic pyrite surfaces using Raman spectroscopy. Our results indicate an important correlation between the evolution of S n 2−/S0 surface species ratio and biofilm formation. Hence, pyrite surfaces with mainly passive-sulfur species were less colonized by A. thiooxidans as compared to surfaces with active sulfur species. These results provide knowledge that may contribute to establishing interfacial conditions that enhance or delay metal sulfide (MS) dissolution, as a function of the biofilm formed by sulfur-oxidizing bacteria.
Keywords: Acidithiobacillus thiooxidans ; Electrooxidation; Reactivity; Pyrite; Sulfur; Biofilms
Impact of an indigenous microbial enhanced oil recovery field trial on microbial community structure in a high pour-point oil reservoir by Fan Zhang; Yue-Hui She; Hua-Min Li; Xiao-Tao Zhang; Fu-Chang Shu; Zheng-Liang Wang; Long-Jiang Yu; Du-Jie Hou (pp. 811-821).
Based on preliminary investigation of microbial populations in a high pour-point oil reservoir, an indigenous microbial enhanced oil recovery (MEOR) field trial was carried out. The purpose of the study is to reveal the impact of the indigenous MEOR process on microbial community structure in the oil reservoir using 16Sr DNA clone library technique. The detailed monitoring results showed significant response of microbial communities during the field trial and large discrepancies of stimulated microorganisms in the laboratory and in the natural oil reservoir. More specifically, after nutrients injection, the original dominant populations of Petrobacter and Alishewanella in the production wells almost disappeared. The expected desirable population of Pseudomonas aeruginosa, determined by enrichment experiments in laboratory, was stimulated successfully in two wells of the five monitored wells. Unexpectedly, another potential population of Pseudomonas pseudoalcaligenes which were not detected in the enrichment culture in laboratory was stimulated in the other three monitored production wells. In this study, monitoring of microbial community displayed a comprehensive alteration of microbial populations during the field trial to remedy the deficiency of culture-dependent monitoring methods. The results would help to develop and apply more MEOR processes.
Keywords: Oil reservoir; Indigenous microbial enhanced oil recovery (MEOR); Microbial community; Stimulated microorganisms
Erratum to: Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris
by Byung-Kwon Choi; Shannon Warburton; Heping Lin; Rohan Patel; Istvan Boldogh; Michael Meehl; Marc d’Anjou; Liza Pon; Terrance A. Stadheim; Natarajan Sethuraman (pp. 823-823).