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Applied Microbiology and Biotechnology (v.95, #1)

Synthesis and production of unsaturated and polyunsaturated fatty acids in yeast: current state and perspectives by Hiroshi Uemura (pp. 1-12).

Recently, many genes involved in the formation of unsaturated and polyunsaturated fatty acids (PUFAs) were isolated. In most cases, their activities were confirmed by expressing them in the well-studied model organism Saccharomyces cerevisiae because its fatty acid compositions are very simple and it does not contain PUFAs. Taking advantage of its genetic tractability and increasing wealth of accessible data, many groups are attempting to produce various useful fatty acids in the model yeasts, mainly in S. cerevisiae. This review describes typical such examples including a very recent study on the expression of a fatty acid hydroxylase gene in fission yeast Schizosaccharomyces pombe. Furthermore, the impact of the genetically engineered alteration of fatty acid composition on the stress tolerance is presented because unsaturated fatty acids have crucial roles in membrane fluidity and signaling processes. Lastly, recent attempts at increasing lipid content in S. cerevisiae are discussed.

Keywords: S. cerevisiae ; S. pombe ; Polyunsaturated fatty acids; Fatty acid desaturase; Elongase; Fatty acid hydroxylase

Effect of impurities in biodiesel-derived waste glycerol on the performance and feasibility of biotechnological processes by Afroditi Chatzifragkou; Seraphim Papanikolaou (pp. 13-27).

The rapid development of biodiesel production technology has led to the generation of tremendous quantities of glycerol wastes, as the main by-product of the process. Stoichiometrically, it has been calculated that for every 100 kg of biodiesel, 10 kg of glycerol are produced. Based on the technology imposed by various biodiesel plants, glycerol wastes may contain numerous kinds of impurities such as methanol, salts, soaps, heavy metals, and residual fatty acids. This fact often renders biodiesel-derived glycerol unprofitable for further purification. Therefore, the utilization of crude glycerol though biotechnological means represents a promising alternative for the effective management of this industrial waste. This review summarizes the effect of various impurities–contaminants that are found in biodiesel-derived crude glycerol upon its conversion by microbial strains in biotechnological processes. Insights are given concerning the technologies that are currently applied in biodiesel production, with emphasis to the impurities that are added in the composition of crude glycerol, through each step of the production process. Moreover, extensive discussion is made in relation with the impact of the nature of impurities upon the performances of prokaryotic and eukaryotic microorganisms, during crude glycerol bioconversions into a variety of high added-value metabolic products. Finally, aspects concerning ways of crude glycerol treatment for the removal of inhibitory contaminants as reported in the literature are given and comprehensively discussed.

Keywords: Crude glycerol; Biodiesel; Impurities; Biotechnological conversions; Fermentations

Impact of plant derivatives on the growth of foodborne pathogens and the functionality of probiotics by Rabin Gyawali; Salam A. Ibrahim (pp. 29-45).

Numerous studies have been published on the antimicrobial and antioxidant properties of various plant components. However, there is relatively little information on the impact of such components on the enhancement of probiotics and production of antimicrobial compounds from these probiotics. Hence, this paper focuses on the influence of plant-derived components against pathogens, enhancement of cell viability and functionality of probiotics, and potential applications of such components in food safety and human health.

Keywords: Plant components; Probiotics; Viability; Pathogens

Endophytic fungi: novel sources of anticancer lead molecules by Sheela Chandra (pp. 47-59).

Cancer is a major killer disease all over the world and more than six million new cases are reported every year. Nature is an attractive source of new therapeutic compounds, as a tremendous chemical diversity is found in millions of species of plants, animals, and microorganisms. Plant-derived compounds have played an important role in the development of several clinically useful anti-cancer agents. These include vinblastine, vincristine, camptothecin, podophyllotoxin, and taxol. Production of a plant-based natural drug is always not up to the desired level. It is produced at a specific developmental stage or under specific environmental condition, stress, or nutrient availability; the plants may be very slow growing taking several years to attain a suitable growth phase for product accumulation and extraction. Considering the limitations associated with the productivity and vulnerability of plant species as sources of novel metabolites, microorganisms serve as the ultimate, readily renewable, and inexhaustible source of novel structures bearing pharmaceutical potential. Endophytes, the microorganisms that reside in the tissues of living plants, are relatively unstudied and offer potential sources of novel natural products for exploitation in medicine, agriculture and the pharmaceutical industry. They develop special mechanisms to penetrate inside the host tissue, residing in mutualistic association and their biotransformation abilities opens a new platform for synthesis of novel secondary metabolites. They produce metabolites to compete with the epiphytes and also with the plant pathogens to maintain a critical balance between fungal virulence and plant defense. It is therefore necessary that the relationship between the plants and endophytes during the accumulation of these secondary metabolites is studied. Insights from such research would provide alternative methods of natural product drug discovery which could be reliable, economical, and environmentally safe.

Keywords: Endophytes; Podophyllotoxin; Taxol; Vinca alkaloids; Camptothecin

Transcriptomic studies of phosphate control of primary and secondary metabolism in Streptomyces coelicolor by Juan F. Martín; Fernando Santos-Beneit; Antonio Rodríguez-García; Alberto Sola-Landa; Margaret C. M. Smith; Trond E. Ellingsen; Kay Nieselt; Nigel J. Burroughs; Ellizabeth M. H. Wellington (pp. 61-75).

Phosphate controls the biosynthesis of many classes of secondary metabolites that belong to different biosynthetic groups, indicating that phosphate control is a general mechanism governing secondary metabolism. We refer in this article to the molecular mechanisms of regulation, mediated by the two-component system PhoR–PhoP, of the primary metabolism and the biosynthesis of antibiotics. The two-component PhoR–PhoP system is conserved in all Streptomyces and related actinobacteria sequenced so far, and involves a third component PhoU that modulates the signal transduction cascade. The PhoP DNA-binding sequence is well characterized in Streptomyces coelicolor. It comprises at least two direct repeat units of 11 nt, the first seven of which are highly conserved. Other less conserved direct repeats located adjacent to the core ones can also be bound by PhoP through cooperative protein–protein interactions. The phoR–phoP operon is self-activated and requires phosphorylated PhoP to mediate the full response. About 50 up-regulated PhoP-dependent genes have been identified by comparative transcriptomic studies between the parental S. coelicolor M145 and the ΔphoP mutant strains. The PhoP regulation of several of these genes has been studied in detail using EMSA and DNase I footprinting studies as well as in vivo expression studies with reporter genes and RT-PCR transcriptomic analyses.

Keywords: Streptomyces genetics; Secondary metabolite biosynthesis; PhoP-mediated regulation; Transcriptomic studies; Expression profiles; Phosphate control

Degradation and assimilation of aromatic compounds by Corynebacterium glutamicum: another potential for applications for this bacterium? by Xi-Hui Shen; Ning-Yi Zhou; Shuang-Jiang Liu (pp. 77-89).

With the implementation of the well-established molecular tools and systems biology techniques, new knowledge on aromatic degradation and assimilation by Corynebacterium glutamicum has been emerging. This review summarizes recent findings on degradation of aromatic compounds by C. glutamicum. Among these findings, the mycothiol-dependent gentisate pathway was firstly discovered in C. glutamicum. Other important knowledge derived from C. glutamicum would be the discovery of linkages among aromatic degradation and primary metabolisms such as gluconeogenesis and central carbon metabolism. Various transporters in C. glutamicum have also been identified, and they play an essential role in microbial assimilation of aromatic compounds. Regulation on aromatic degradation occurs mainly at transcription level via pathway-specific regulators, but global regulator(s) is presumably involved in the regulation. It is concluded that C. glutamicum is a very useful model organism to disclose new knowledge of biochemistry, physiology, and genetics of the catabolism of aromatic compounds in high GC content Gram-positive bacteria, and that the new physiological properties of aromatic degradation and assimilation are potentially important for industrial applications of C. glutamicum.

Keywords: Corynebacterium glutamicum ; Aromatic compounds; Degradation and assimilation; Transport; Regulation

Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata by Chen-Sheng Zhang; Zhi-Jun Zhang; Chun-Xiu Li; Hui-Lei Yu; Gao-Wei Zheng; Jian-He Xu (pp. 91-99).

(R)-o-Chloromandelic acid is the key precursor for the synthesis of Clopidogrel®, a best-selling cardiovascular drug. Although nitrilases are often used as an efficient tool in the production of α-hydroxy acids, there is no practical nitrilase specifically developed for (R)-o-chloromandelic acid. In this work, a new nitrilase from Labrenzia aggregata (LaN) was discovered for the first time by genomic data mining, which hydrolyzed o-chloromandelonitrile with high enantioselectivity, yielding (R)-o-chloromandelic acid in 96.5% ee. The LaN was overexpressed in Escherichia coli BL21 (DE3), purified, and its catalytic properties were studied. When o-chloromandelonitrile was used as the substrate, the V _max and K _m of LaN were 2.53 μmol min⁻¹ mg⁻¹ protein and 0.39 mM, respectively, indicating its high catalytic efficiency. In addition, a study of substrate spectrum showed that LaN prefers to hydrolyze arylacetonitriles. To relieve the substrate inhibition and to improve the productivity of LaN, a biphasic system of toluene–water (1:9, v/v) was adopted, in which o-chloromandelonitrile of 300 mM (apparent concentration, based on total volume) could be transformed by LaN in 8 h, giving an isolated yield of 94.5%. The development of LaN makes it possible to produce (R)-o-chloromandelic acid by deracemizing o-chloromandelonitrile with good ee value and high substrate concentration.

Keywords: Nitrilase; Labrenzia aggregate ; Enantioselective hydrolysis; (R)-o-Chloromandelic acid; Biphasic system

Production and characterisation of recombinant α-l-arabinofuranosidase for production of xylan hydrogels by A. F. A. Chimphango; S. H. Rose; W. H. van Zyl; J. F. Görgens (pp. 101-112).

A recombinant strain of the protease-deficient, non-acidifying pH mutant Aspergillus niger D15 (A. niger D15 [abfB]) strain was developed to secrete α-l-arabinofuranosidase (AbfB) free of endo-1,4-β-xylanases for selective hydrolysis of xylan into hydrogels. The A. niger D15 [abfB] strain expressed the α-l-arabinofuranosidase abfB gene under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd P) and glucoamylase terminator (glaA T) in fermentation cultures containing 10 % glucose. The yield, activity, purity, kinetics and ability of the recombinant AbfB to selectively hydrolyse xylans into hydrogels were assessed. The recombinant AbfB secreted in 125-mL shake flasks and 10-L bioreactor fermentation cultures had specific activities against ρ-nitrophenyl-α-arabinofuranoside of up to 4.4 and 2.7 U g⁻¹ (dry weight), respectively. In addition, the recombinant AbfB was present as a single protein species on silver-stained 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The recombinant AbfB had optimal activity at 40–55 °C and pH 3.0 to pH 5.0 and was stable at temperature and pH of up to 60 °C and pH 6.0, respectively. About 20 % of the available arabinose in the xylan was released by the recombinant AbfB from the hydrolysis of low viscosity wheat and oat spelt arabinoxylans and about 9 and 5 % from bagasse and bamboo arabinoglucuronoxylans, respectively, that led to the formation of the hydrogels. Therefore, the constructed A. niger D15 [abfB] strain presented a microbial system for the production of recombinant AbfB with the required purity for the modification of xylans into hydrogels.

Keywords: Aspergillus niger ; α-l-Arabinofuranosidase; Xylan; Arabinose; Hydrogels

The echinocandin B producer fungus Aspergillus nidulans var. roseus ATCC 58397 does not possess innate resistance against its lipopeptide antimycotic by Viktória Tóth; Csilla Terézia Nagy; István Pócsi; Tamás Emri (pp. 113-122).

Aspergillus nidulans var. roseus ATCC 58397 is an echinocandin B (ECB) producer ascomycete with great industrial importance. As demonstrated by ECB/caspofungin sensitivity assays, A. nidulans var. roseus does not possess any inherent resistance to echinocandins, and its tolerance to these lipopeptide antimycotics are even lower than those of the non-producer A. nidulans FGSC A4 strain. Under ECB producing conditions or ECB exposures, A. nidulans var. roseus induced its ECB tolerance via up-regulating elements of the chitin biosynthetic machinery and, hence, through changing dynamically the composition of its own cell wall. Importantly, although the specific β-1,3-glucan synthase activity was elevated, these changes reduced the β-glucan content of hyphae considerably, but the expression of fksA, encoding the catalytic subunit of β-1,3-glucan synthase, the putative target of echinocandins in the aspergilli, was not affected. These data suggest that compensatory chitin biosynthesis is the centerpiece of the induced ECB tolerance of A. nidulans var. roseus. It is important to note that the induced tolerance to ECB (although resulted in paradoxical growth at higher ECB concentrations) was accompanied with reduced growth rate and, under certain conditions, even sensitized the fungus to other stress-generating agents like SDS. We hypothesize that although ECB-resistant mutants may arise in vivo in A. nidulans var. roseus cultures, their widespread propagation is severely restricted by the disadvantageous physiological effects of such mutations.

Keywords: Echinocandin resistance; Aspergillus nidulans var. roseus ; Emericella rugulosa ; Paradoxical growth; Chitin synthesis

CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase by Thuy T. B. Ly; Yogan Khatri; Josef Zapp; Michael C. Hutter; Rita Bernhardt (pp. 123-133).

Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and β-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and β-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-β-ionone and 13-hydroxy-nootkatone, were confirmed by ¹H and ¹³C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the K m and k _cat values were calculated. The results of docking α-/β-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations.

Keywords: Cytochrome P450; Terpenoid; Nootkatone; Ionone; CYP264B1; Sorangium cellulosum So ce56

Metagenomic cellulases highly tolerant towards the presence of ionic liquids—linking thermostability and halotolerance by Nele Ilmberger; Diana Meske; Julia Juergensen; Michael Schulte; Peter Barthen; Ulrich Rabausch; Angel Angelov; Markus Mientus; Wolfgang Liebl; Ruth A. Schmitz; Wolfgang R. Streit (pp. 135-146).

Cellulose is an important renewable resource for the production of bioethanol and other valuable compounds. Several ionic liquids (ILs) have been described to dissolve water-insoluble cellulose and/or wood. Therefore, ILs would provide a suitable reaction medium for the enzymatic hydrolysis of cellulose if cellulases were active and stable in the presence of high IL concentrations. For the discovery of novel bacterial enzymes with elevated stability in ILs, metagenomic libraries from three different hydrolytic communities (i.e. an enrichment culture inoculated with an extract of the shipworm Teredo navalis, a biogas plant sample and elephant faeces) were constructed and screened. Altogether, 14 cellulolytic clones were identified and subsequently assayed in the presence of six different ILs. The most promising enzymes, CelA2, CelA3 (both derived from the biogas plant) and CelA84 (derived from elephant faeces), showed high activities (up to 6.4 U/mg) in the presence of 30% (v/v) ILs. As these enzymes were moderately thermophilic and halotolerant, they retained 40% to 80% relative activity after 34 days in 4 M NaCl, and they were benchmarked with two thermostable enzymes, CelA from Thermotoga maritima and Cel5K from a metagenome library derived from Avachinsky crater in Kamchatka. These enzymes also exhibited high activity (up to 11.1 U/mg) in aqueous IL solutions (30% (v/v)). Some of the enzymes furthermore exhibited remarkable stability in 60% (v/v) IL. After 4 days, CelA3 and Cel5K retained up to 79% and 100% of their activity, respectively. Altogether, the obtained data suggest that IL tolerance appears to correlate with thermophilicity and halotolerance.

Keywords: Cellulase; Ionic liquid; Metagenomics; Halotolerance; Thermostability

Novel magnetic microspheres of P (GMA-b-HEMA): preparation, lipase immobilization and enzymatic activity in two phases by Yanjun Cui; Xia Chen; Yanfeng Li; Xiao Liu; Lin Lei; Sunting Xuan (pp. 147-156).

Magnetic oleic-acid-coated Fe₃O₄ nanoparticles were first introduced into 1, 1-diphenylethylene (DPE)-controlled radical polymerization system to prepare superparamagnetic microspheres for enzyme immobilization by two steps of polymerization. In the presence of DPE, glycidyl methacrylate, 2-hydroxyethyl methacrylate and methacryloxyethyl trimethyl ammonium chloride with charge were selected as copolymering monomers based on their reactive functional group and excellent biocompatibility which were suitable for immobilization of Candida rugosa lipase (CRL). The resulting magnetic microspheres were characterized by means of scanning electron microscope, Fourier transform infrared spectrum, thermogravimetric analysis and vibrating sample magnetometry. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE analysis was also conducted to demonstrate whether CRL is covalently immobilized or only physically adsorbed. The results indicated that the polymerization was successfully carried out, and lipase was immobilized on the magnetic microspheres through ionic adsorption and covalent binding under mild conditions. The immobilized lipase exhibited high activity recovery (69.7%), better resistance to pH and temperature inactivation in aqueous phase, as well as superior reusability in nonaqueous phase. The data showed that the resulting carrier could hold an amphiphilic property.

Keywords: DPE (1; 1-diphenylethylene); Block copolymer; Lipase Immobilization; Enzymatic activity

Structural analysis and biosynthetic engineering of a solubility-improved and less-hemolytic nystatin-like polyene in Pseudonocardia autotrophica by Mi-Jin Lee; Dekun Kong; Kyuboem Han; David H. Sherman; Linquan Bai; Zixin Deng; Shuangjun Lin; Eung-Soo Kim (pp. 157-168).

Polyene antibiotics such as nystatin are a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain an approximately 125.7-kb region of contiguous DNA with a total of 23 open reading frames, which are involved in the biosynthesis and regulation of a structurally unique polyene natural product named NPP. Here, we report the complete structure of NPP, which contains an aglycone identical to nystatin and harbors a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine. A mutant generated by inactivation of a sole glycosyltransferase gene (nppDI) within the npp gene cluster can be complemented in trans either by nppDI-encoded protein or by its nystatin counterpart, NysDI, suggesting that the two sugars might be attached by two different glycosyltransferases. Compared with nystatin (which bears a single sugar moiety), the di-sugar containing NPP exhibits approximately 300-fold higher water solubility and 10-fold reduced hemolytic activity, while retaining about 50% antifungal activity against Candida albicans. These characteristics reveal NPP as a promising candidate for further development into a pharmacokinetically improved, less-cytotoxic polyene antifungal antibiotic.

Keywords: Polyene; Antifungal; Nystatin; Pseudonocardia ; Less-hemolytic

Improving putrescine production by Corynebacterium glutamicum by fine-tuning ornithine transcarbamoylase activity using a plasmid addiction system by Jens Schneider; Dorit Eberhardt; Volker F. Wendisch (pp. 169-178).

Corynebacterium glutamicum shows a great potential for the production of the polyamide monomer putrescine (1,4-diaminobutane). Previously, we constructed the putrescine-producing strain PUT1 by deletion of argF, the gene for ornithine transcarbamoylase (OTC), and argR, encoding the l-arginine repressor, combined with heterologous expression of the Escherichia coli gene for l-ornithine decarboxylase SpeC. As a consequence of argF deletion, this strain requires supplementation of l-arginine and shows growth-decoupled putrescine production. To avoid costly supplementation with l-arginine and the strong feedback inhibition of the key enzyme N-acetylglutamate kinase (ArgB) by l-arginine, a plasmid addiction system for low-level argF expression was developed. By fine-tuning argF expression through modifications of the promoter, the translational start codon and/or the ribosome binding site, high productivity and titer could be obtained. OTC activity varied almost thousandfold between 960 and 1 mU mg⁻¹ resulting in putrescine yields on glucose from less than 0.001 up to 0.26 g g⁻¹, the highest yield in bacteria reported to date. The most promising strain, designated PUT21, was characterized comprehensively. PUT21 strain grew with a rate of 0.19 h⁻¹ in mineral salt medium without the need for l-arginine supplementation and produced putrescine with a yield of 0.16 g g⁻¹ glucose at a volumetric productivity of 0.57 g L⁻¹ h⁻¹ and a specific productivity of 0.042 g g⁻¹ h⁻¹. The carbon balance suggested that no major unidentified by-product was produced. Compared to the first-generation strain PUT1, the putrescine yield observed with PUT21 was increased by 60%. In fed-batch cultivation with C. glutamicum PUT21, a putrescine titer of 19 g L⁻¹ at a volumetric productivity of 0.55 g L⁻¹ h⁻¹ and a yield of 0.16 g g⁻¹ glucose could be achieved. Moreover, while plasmid segregation of the initial strain required antibiotic selection, plasmid segregation in C. glutamicum PUT21 was fully stable for more than 60 generations without antibiotic selection even in the presence of l-arginine. The ornithine decarboxylase gene speC was expressed from this argF addiction plasmid ensuring stable putrescine production by the engineered C. glutamicum strain.

Keywords: Corynebacterium glutamicum ; Metabolic engineering; Plasmid addiction system; Putrescine; 1,4-Diaminobutane; Diamine; Polyamine; Polyamide

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe by Roman Holic; Hisashi Yazawa; Hiromichi Kumagai; Hiroshi Uemura (pp. 179-187).

In an effort to produce ricinoleic acid (12-hydroxy-octadeca-cis-9-enoic acid: C18:1-OH) as a petrochemical replacement in a variety of industrial processes, we introduced Claviceps purpurea oleate ∆12-hydroxylase gene (CpFAH12) to Schizosaccharomyces pombe, putting it under the control of inducible nmt1 promoter. Since Fah12p is able to convert oleic acid to ricinoleic acid, we thought that S. pombe, in which around 75% of total fatty acid (FA) is oleic acid, would accordingly be an ideal microorganism for high production of ricinoleic acid. Unfortunately, at the normal growth temperature of 30 °C, S. pombe cells harboring CpFAH12 grew poorly when the CpFAH12 gene expression was induced, perhaps implicating ricinoleic acid as toxic in S. pombe. However, in line with a likely thermoinstability of Fah12p, there was almost no growth inhibition at 37 °C or, by contrast with 30 °C and lower temperatures, ricinoleic acid accumulation. Accordingly, various optimization steps led to a regime with preliminary growth at 37 °C followed by a 5-day incubation at 20 °C, and the level of ricinoleic acid reached 137.4 μg/ml of culture that corresponded to 52.6% of total FA.

Keywords: Schizosaccharomyces pombe ; Yeast; Ricinoleic acid; FAH12 ; Fatty acid hydroxylase; Fatty acids

Transcriptome profiling of Zymomonas mobilis under furfural stress by Ming-xiong He; Bo Wu; Zong-xia Shui; Qi-chun Hu; Wen-guo Wang; Fu-rong Tan; Xiao-yu Tang; Qi-li Zhu; Ke Pan; Qing Li; Xiao-hong Su (pp. 189-199).

Furfural from lignocellulosic hydrolysates is the prevalent inhibitor to microorganisms during cellulosic ethanol production, but the molecular mechanisms of tolerance to this inhibitor in Zymomonas mobilis are still unclear. In this study, genome-wide transcriptional responses to furfural were investigated in Z. mobilis using microarray analysis. We found that 433 genes were differentially expressed in response to furfural. Furfural up- or down-regulated genes related to cell wall/membrane biogenesis, metabolism, and transcription. However, furfural has a subtle negative effect on Entner–Doudoroff pathway mRNAs. Our results revealed that furfural had effects on multiple aspects of cellular metabolism at the transcriptional level and that membrane might play important roles in response to furfural. This research has provided insights into the molecular response to furfural in Z. mobilis, and it will be helpful to construct more furfural-resistant strains for cellulosic ethanol production.

Keywords: Furfural; Lignocellulosic hydrolysates; Bioethanol; Zymomonas mobilis ; Microarray

Phosphoproteomic investigation of a solvent producing bacterium Clostridium acetobutylicum by Xue Bai; Zhihong Ji (pp. 201-211).

In this study, we employed TiO₂ enrichment and high accuracy liquid chromatography-mass spectrometry-mass spectrometry to identify the phosphoproteome of Clostridium acetobutyicum ATCC824 in acidogenesis and solventogenesis. As many as 82 phosphopeptides in 61 proteins, with 107 phosphorylated sites on serine, threonine, or tyrosine, were identified with high confidence. We detected 52 phosphopeptides from 44 proteins in acidogenesis and 70 phosphopeptides from 51 proteins in solventogenesis, respectively. Bioinformatic analysis revealed most of the phosphoproteins located in cytoplasm and participated in carbon metabolism. Based on comparison between the two stages, we found 27 stage-specific phosphorylated proteins (10 in acidogenesis and 17 in solventogenesis), some of which were solvent production-related enzymes and metabolic regulators, showed significantly different phosphorylated status. Further analysis indicated that protein phosphorylation could be involved in the shift of stages or in solvent production pathway directly. Comparison against several other organisms revealed the evolutionary diversity among them on phosphorylation level in spite of their high homology on protein sequence level.

Keywords: Phosphoproteome; Mass spectrometry; Clostridium acetobutylicum ; Biological solvent production

Controlled synthesis of Mn₂O₃ nanowires by hydrothermal method and their bactericidal and cytotoxic impact: a promising future material by M. Shamshi Hassan; Touseef Amna; Dipendra Raj Pandeya; A. M. Hamza; Yang You Bing; Hyun-Chel Kim; Myung-Seob Khil (pp. 213-222).

Mn₂O₃ nanowires with diameter ~70 nm were synthesized by a simple hydrothermal method using Mn(II) nitrate as precursor. X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy techniques were employed to study structural features and chemical composition of the synthesized nanowires. A biological evaluation of the antimicrobial activity and cytotoxicity of Mn₂O₃ nanowires was carried out using Escherichia coli and mouse myoblast C₂C₁₂ cells as model organism and cell lines, respectively. The antibacterial activity and the acting mechanism of Mn₂O₃ nanowires were investigated by using growth inhibition studies and analyzing the morphology of the bacterial cells following the treatment with nanowires. These results suggest that the pH is critical factor affecting the morphology and production of the Mn₂O₃ nanowires. Method developed in the present study provided optimum production of Mn₂O₃ nanowires at pH ~ 9. The Mn₂O₃ nanowires showed significant antibacterial activity against the E. coli strain, and the lowest concentration of Mn₂O₃ nanowires solution inhibiting the growth of E. coli was found to be 12.5 μg/ml. TEM analysis demonstrated that the exposure of the selected microbial strains to the nanowires led to disruption of the cell membranes and leakage of the internal contents. Furthermore, the cytotoxicity results showed that the inhibition of C₂C₁₂ increases with the increase in concentration of Mn₂O₃ nanowires. Our results for the first time highlight the cytotoxic and bactericidal potential of Mn₂O₃ nanowires which can be utilized for various biomedical applications.

Keywords: Mn₂O₃ nanowires; Hydrothermal method; Antibacterial activity; E. coli ; Cytotoxicity

Modeling of permeabilization process in Pseudomonas putida G7 for enhanced limonin bioconversion by Meenakshi Malik; Abhijit Ganguli; Moushumi Ghosh (pp. 223-231).

A facile process of enhanced whole cell biotransformation to debitter the triterpenoid limonin in citrus juices was optimized in this work. To maximize bioconversion, permeabilization conditions were modeled using response surface methodology. A central composite rotatable design with four significant variables (concentration, temperature, pH, and treatment time) was employed. The second order polynomial equations with R ² values above 0.9 showed good correspondence between experimental and predicted values. The concentration, temperature, pH, and treatment time as well as their interactions had significant effects (p < 0.001) on limonin bioconversion. The optimum operating conditions for permeabilization were observed at a Na₂EDTA concentration of 1.5 μM, treatment time of 15 min, temperature of 28 °C, and pH 8. A maximum reduction of 76.71% in the limonin content was achieved within 150 min under selected conditions. The results are promising for refining permeabilization technique for whole cell biocatalysts thereby improving the debittering of citrus juices significantly.

Keywords: Response surface methodology (RSM); Limonin; Permeabilization; Pseudomonas putida G7; Debittering

Application of purified recombinant antigenic spike fragments to the diagnosis of avian infectious bronchitis virus infection by Kuan-Hsun Lin; Chuen-Fu Lin; Shyan-Song Chiou; Ai-Ping Hsu; Min-Shiuh Lee; Chao-Chin Chang; Tien-Jye Chang; Jui-Hung Shien; Wei-Li Hsu (pp. 233-242).

The spike (S) protein, containing two subunits, S1 and S2, is the major immunity-eliciting antigen of avian infectious bronchitis virus (IBV), a highly contagious disease of chickens. Several immunogenic regions, mainly located within the S1 subunit, have been identified. Nonetheless, these immune-dominant regions were defined using selected monoclonal antibodies or using a short peptide approach that involves only certain limited regions of the S protein. In addition, some immune-dominant regions are located in hypervariable regions (HVRs) which are not present in all serotypes. Hence, the aim of this study was to determine a broader range of antigenic regions that have strong antibody eliciting ability; these could then be applied for development of an IBV-diagnostic tool. Initially, the S1 and part of the S2 subunit protein (24–567 amino acids) were expressed as five fragments in prokaryotic system. The antigenicity was confirmed using IBV immunized sera. Performance of the S subfragments was evaluated by ELISA using a panel of field chicken sera with known IBV titres determined by a commercial kit. This indicated that, among the five antigenic recombinant proteins, the region S-E showed the highest specificity and sensitivity, namely 95.38 % and 96.29 %, respectively. The κ value for the in-house ELISA using the S-E fragment compared to a commercial kit was 0.9172, indicating a high agreement between these two methods. As region S-E harbors strong immunogenicity within the spike protein, it has the potential to be exploited as an antigen when developing a cost-effective ELISA-based diagnosis tool.

Keywords: Infectious bronchitis virus; Spike protein; Antigenic regions; ELISA

Effect of ZnO and TiO₂ nanoparticles preilluminated with UVA and UVB light on Escherichia coli and Bacillus subtilis by Shin Woong Kim; Youn-Joo An (pp. 243-253).

We evaluated the effects of zinc oxide (ZnO) and titanium dioxide (TiO₂) nanoparticles (NPs) preilluminated with ultraviolet light on Escherichia coli and Bacillus subtilis. The experiments were conducted using three different types of light: visible, Ultraviolet A (UVA, 315–400 nm), and Ultraviolet B (UVB, 280–315 nm). The bacteria were exposed to NPs, either as liquid suspensions for growth inhibition assays or on agar plates for colony forming unit (CFU) assays. We found that the ZnO NPs were more toxic when preilluminated with UVA or UVB light than with visible light in both growth inhibition and CFU assays. TiO₂ NPs were not toxic to the bacteria under UVA or UVB preillumination conditions. The photo-dissolution of ZnO NPs increased with UV preillumination, which could explain the observed toxicity of ZnO NPs. We detected oxidative stress elicited by photoactive nanoparticles by measuring superoxide dismutase activity. The results of this study show that the toxicity of photoactive nanoparticles can be increased by UV preillumination by dissolution of toxic ions, which suggests the potential for preillumination-dependent toxicity of nanoparticles on soil environments in low light or darkness.

Keywords: Bacillus subtilis ; Escherichia coli ; Microbial assay; TiO₂ nanoparticle; UV preillumination; ZnO nanoparticle

Isolation and characterization of a Klebsiella oxytoca strain for simultaneous azo-dye anaerobic reduction and bio-hydrogen production by Lei Yu; Wen-Wei Li; Michael Hon-Wah Lam; Han-Qing Yu; Chao Wu (pp. 255-262).

A facultative anaerobic bacteria strain GS-4-08, isolated from an anaerobic sequence batch reactor for synthetic dye wastewater treatment, was investigated for azo-dye decolorization. This bacterium was identified as a member of Klebsiella oxytoca based on Gram staining, morphology characterization and 16S rRNA gene analysis. It exhibited a good capacity of simultaneous decolorization and hydrogen production in the presence of electron donor. The hydrogen production was less affected even at a high Methyl Orange (MO) concentration of 0.5 mM, indicating a superior tolerability of this strain to MO. This efficient bio-hydrogen production from electron donor can not only avoid bacterial inhibition due to accumulation of volatile fatty acids during MO decolorization, but also can recover considerable energy from dye wastewater.

Keywords: Anaerobic; Bio-hydrogen; Decolorization; Electron donor; Klebisiella oxytoca ; Methyl Orange (MO)

2,4-DNT removal in intimately coupled photobiocatalysis: the roles of adsorption, photolysis, photocatalysis, and biotransformation by Donghui Wen; Guozheng Li; Rui Xing; Seongjun Park; Bruce E. Rittmann (pp. 263-272).

The removal of 2,4-dinitrotoluene (2,4-DNT) by simultaneous UV-photo(cata)lysis and biodegradation was explored using intimately coupled photolysis/photocatalysis and biodegradation (ICPB) with two novel porous carriers. First, a porous ceramic carrier was used to attach the photocatalyst (TiO₂) on its exterior and accumulate biomass in its interior. UV irradiation alone decomposed 71% of the 2,4-DNT in 60 h, and TiO₂ catalyst improved the photolysis to 77%. Second, a macroporous sponge carrier was used to strongly adsorb 2,4-DNT and protect microorganisms from 2,4-DNT inhibition and UV irradiation. The main photolytic reactions were reduction of the nitryl to amino and hydrolysis of the amino to release NH ₄ ⁺ . The main biodegradation reactions were oxidative release of NO ₃ ⁻ and accelerated reductive release of NH ₄ ⁺ . ICPB more thoroughly released inorganic N, with nearly equal amounts being oxidized to nitrate and reduced to ammonium. The genera Burkholderia and Bacillus were found inside the sponge carriers, and they are associated with biodegradation of DNT and its photolysis intermediates. Therefore, using an adsorbent and macroporous biofilm carrier enabled the effective removal of 2,4-DNT by ICPB.

Keywords: 2,4-DNT; Biodegradation; Photolysis; Adsorption; Intimate coupling

Erratum to: Development and characterization of DehaloR^2, a novel anaerobic microbial consortium performing rapid dechlorination of TCE to ethene by Michal Ziv-El; Anca G. Delgado; Ying Yao; Dae-Wook Kang; Katherine G. Nelson; Rolf U. Halden; Rosa Krajmalnik-Brown (pp. 273-274).

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Applied Microbiology and Biotechnology (v.95, #1)

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Applied Microbiology and Biotechnology (v.95, #1)