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Applied Microbiology and Biotechnology (v.94, #2)

Role of nanotechnology in agriculture with special reference to management of insect pests by Mahendra Rai; Avinash Ingle (pp. 287-293).

Nanotechnology is a promising field of interdisciplinary research. It opens up a wide array of opportunities in various fields like medicine, pharmaceuticals, electronics and agriculture. The potential uses and benefits of nanotechnology are enormous. These include insect pests management through the formulations of nanomaterials-based pesticides and insecticides, enhancement of agricultural productivity using bio-conjugated nanoparticles (encapsulation) for slow release of nutrients and water, nanoparticle-mediated gene or DNA transfer in plants for the development of insect pest-resistant varieties and use of nanomaterials for preparation of different kind of biosensors, which would be useful in remote sensing devices required for precision farming. Traditional strategies like integrated pest management used in agriculture are insufficient, and application of chemical pesticides like DDT have adverse effects on animals and human beings apart from the decline in soil fertility. Therefore, nanotechnology would provide green and efficient alternatives for the management of insect pests in agriculture without harming the nature. This review is focused on traditional strategies used for the management of insect pests, limitations of use of chemical pesticides and potential of nanomaterials in insect pest management as modern approaches of nanotechnology.

Keywords: Nano-pesticide; Sustainable agriculture; Precision farming; Chemical pesticide

Glycosaminoglycan polysaccharide biosynthesis and production: today and tomorrow by Paul L. DeAngelis (pp. 295-305).

Glycosaminoglycans [GAGs] are essential heteropolysaccharides in vertebrate tissues that are also, in certain cases, employed as virulence factors by microbes. Hyaluronan [HA], heparin, and chondroitin sulfate [CS] are GAGs currently used in various medical applications and together are multi-billion dollar products thus targets for production by animal-free manufacture. By using bacteria as the source of GAGs, the pathogen’s sword may be converted into a plowshare to help avoid potential liabilities springing from the use of animal-derived GAGs including adventitious agents (e.g., prions, pathogens), antigenicity, degradation of the environment, and depletion of endangered species. HA from microbes, which have a chemical structure identical to human HA, has already been commercialized and sold at the ton-scale. Substantial progress towards microbial heparin and CS has been made, but these vertebrate polymers are more complicated structurally than the unsulfated bacterial polysaccharide precursors thus require additional processing steps. This review provides an overview of GAG structure, medical applications, microbial biosynthesis, and the state of bacterial GAG production systems. Representatives of all glycosyltransferase enzymes that polymerize the sugar chains of the three main GAGs have been identified and serve as the core technology to harness, but the proteins involved in sugar precursor formation and chain export steps of biosynthesis are also essential to the GAG production process. In addition, this review discusses future directions and potential important issues. Overall, this area is poised to make great headway to produce safer (both increased purity and more secure supply chains) non-animal GAG-based therapeutics.

Keywords: Polysaccharide; Capsule; Synthase; Glycosyltransferase; Hyaluronan; Heparin; Chondroitin

Microbial heparin/heparan sulphate lyases: potential and applications by C. K. M. Tripathi; Jaspreet Banga; Vikas Mishra (pp. 307-321).

Heparin/heparan sulphate glycosaminoglycans (HSGAGs) are composed of linear chains of 20–100 disaccharide units of N-acetylated d-glucosamine α (1–4) linked to glucuronic acid. HSGAGs are widely distributed on the cell surface and extracellular cell matrix of virtually every mammalian cell type and play critical role in regulating numerous functions of blood vessel wall, blood coagulation, inflammation response and cell differentiation. These glycosaminoglycans present in this extracellular environment very significantly influence the blood coagulation system and cardiovascular functions. Recent studies have investigated the mechanism by which cancer causes thrombosis and emphasizes the importance of the coagulation system in angiogenesis and tumour metastasis. Heparan sulphate/heparin lyases or heparinases are a class of enzymes that are capable of specifically cleaving the (1–4) glycosidic linkages in heparin and heparan sulphate to generate biologically active oligosaccharides with substantially significant and distinct clinical, pharmaceutical and prophylactic/therapeutic applications. Bioavailability and pharmacokinetic behaviour and characteristics of these oligosaccharides vary significantly depending on the origin/nature of the substrate (heparin or heparan sulphate-like glycosaminoglycans), the source of enzyme and method of preparation. Various microorganisms are reported/patented to produce these enzymes with different properties. Heparinases are commercially used for the depolymerization of unfractionated heparin to produce low molecular weight heparins (LMWHs), an effective anticoagulant. Individual LMWHs are chemically different and unique and thus cannot be interchanged therapeutically. Heparinases and LMWHs are reported to control angiogenesis and metastasis also. This review catalogues the degradation of HSGAGs by microbial heparin/heparan sulphate lyases and their potential either specific to the enzymes or with the dual role for generation of oligosaccharides for a new generation of compounds, as shown by various laboratory or clinical studies.

Keywords: Glycosaminoglycans; Heparin lyases; Low molecular weight heparins (LMWHs); Neovascularization; Unfractionated heparin (UFH)

Peculiarities of brown-rot fungi and biochemical Fenton reaction with regard to their potential as a model for bioprocessing biomass by Valdeir Arantes; Jody Jellison; Barry Goodell (pp. 323-338).

This work reviews the brown-rot fungal biochemical mechanism involved in the biodegradation of lignified plant cell walls. This mechanism has been acquired as an apparent alternative to the energetically expensive apparatus of lignocellulose breakdown employed by white-rot fungi. The mechanism relies, at least in the incipient stage of decay, on the oxidative cleavage of glycosidic bonds in cellulose and hemicellulose and the oxidative modification and arrangement of lignin upon attack by highly destructive oxygen reactive species such as the hydroxyl radical generated non-enzymatically via Fenton chemistry $$ ({ ext{F}}{{ ext{e}}^{{{3} + }}} + {{ ext{H}}_{{2}}}{{ ext{O}}_{{2}}} o { ext{F}}{{ ext{e}}^{{{2} + }}} + cdot { ext{OH}}{{ + }^{ - }}{ ext{OH}}) $$ . Modifications in the lignocellulose macrocomponents associated with this non-enzymatic attack are believed to aid in the selective, near-complete removal of polysaccharides by an incomplete cellulase suite and without causing substantial lignin removal. Utilization of this process could provide the key to making the production of biofuel and renewable chemicals from lignocellulose biomass more cost-effective and energy efficient. This review highlights the unique features of the brown-rot fungal non-enzymatic, mediated Fenton reaction mechanism, the modifications to the major plant cell wall macrocomponents, and the implications and opportunities for biomass processing for biofuels and chemicals.

Keywords: Brown-rot fungi; Fenton reaction; Bioconversion; Cellulose degradation; Lignin modification; Oxygen free radicals

Expression and regulation of genes encoding lignocellulose-degrading activity in the genus Phanerochaete by Jacqueline MacDonald; Hitoshi Suzuki; Emma R. Master (pp. 339-351).

As white-rot basidiomycetes, Phanerochaete species are critical to the cycling of carbon sequestered as woody biomass, and are predicted to encode many enzymes that can be harnessed to promote the conversion of lignocellulose to sugars for fermentation to fuels and chemicals. Advances in genomic, transcriptomic, and proteomic technologies have enabled detailed analyses of different Phanerochaete species and have revealed numerous enzyme families required for lignocellulose utilization, as well as insight into the regulation of corresponding genes. Recent studies of Phanerochaete are also exemplified by molecular analyses following cultivation on different wood preparations, and show substrate-dependent responses that were difficult to predict using model compounds or isolated plant polysaccharides. The aim of this mini-review is to synthesize results from studies that have applied recent advances in molecular tools to evaluate the expression and regulation of proteins that contribute to lignocellulose conversion in Phanerochaete species. The identification of proteins with as yet unknown function are also highlighted and noted as important targets for future investigation of white-rot decay.

Keywords: Phanerochaete carnosa ; Fungal genomics; Fungal transcriptomics; Fungal proteomics; Carbohydrate-active enzymes; Ligninolytic enzymes; Lignocellulose bioconversion; White-rot

Evaluation of aroma active compounds in Tuber fruiting bodies by gas chromatography–olfactometry in combination with aroma reconstitution and omission test by Rui-Sang Liu; Dao-Cheng Li; Hong-Mei Li; Ya-Jie Tang (pp. 353-363).

The aroma active compounds of three Tuber fruiting bodies (i.e., Tuber himalayense, Tuber indicum, and Tuber sinense) were firstly systematically evaluated by instrumental gas chromatography–olfactometry combining with quantitative analysis, aroma reconstitution, and omission tests. Twelve aroma active compounds were characterized by aroma extract dilution analysis, and 3-(methylthio) propanal, 3-methylbutanal, and 1-octen-3-ol with the highest flavor dilution (FD) factor (i.e., 1,024–2,048) were suggested as key contributors to the aroma. Odor activity value (OAV) was employed to determine the relative contribution of each compound to the aroma, and the compound with the highest FD factor also had the highest OAV (i.e., 10,234–242,951). Then, the synthetic blends of odorants (aroma reconstitution) were prepared with OAV larger than 15, and their aromas were very similar to the originals. Omission tests were carried out to verify the significance of 3-(methylthio) propanal, 1-octen-3-ol, and 3-methylbutanal as key compounds in the aroma of tested Tuber fruiting bodies.

Keywords: Truffle; Tuber fruiting bodies; Aroma active compounds; Gas chromatography–olfactometry; Aroma reconstitution; Omission test

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from Pseudomonas sp. SG4502 with acquired LA-polymerizing activity by Kenji Tajima; Xuerong Han; Yasuharu Satoh; Ayako Ishii; Yuji Araki; Masanobu Munekata; Seiichi Taguchi (pp. 365-376).

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1_SG and PhaC2_SG). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1_SG to evaluate the potential of the resulting protein as a “thermostable LPE”. The mutated PhaC1_SG [PhaC1_SG(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1_SG(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).

Keywords: Polyhydroxyalkanoate (PHA); Thermal stability; Block copolymer; LA-polymerizing enzyme (LPE); In vitro polymerization; Pseudomonas sp. SG4502

Enzymatic biotransformation of ginsenoside Rb1 to 20(S)-Rg3 by recombinant β-glucosidase from Microbacterium esteraromaticum by Lin-Hu Quan; Jin-Woo Min; Dong-Uk Yang; Yeon-Ju Kim; Deok-Chun Yang (pp. 377-384).

Microbacterium esteraromaticum was isolated from ginseng field. The β-glucosidase gene (bgp1) from M. esteraromaticum was cloned and expressed in Escherichia coli BL21 (DE3). The bgp1 gene consists of 2,496 bp encoding 831 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. The recombinant β-glucosidase enzyme (Bgp1) was purified and characterized. The molecular mass of purified Bgp1 was 87.5 kDa, as determined by SDS-PAGE. Using 0.1 mg ml⁻¹ enzyme in 20 mM sodium phosphate buffer at 37°C and pH 7.0, 1.0 mg ml⁻¹ ginsenoside Rb1 was transformed into 0.444 mg ml⁻¹ ginsenoside Rg3 within 6 h. The Bgp1 sequentially hydrolyzed the outer and inner glucose attached to the C-20 position of ginsenosides Rb1. Bgp1 hydrolyzed the ginsenoside Rb1 along the following pathway: Rb1 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 using the recombinant β-glucosidase.

Keywords: Biotransformation; β-glucosidase; Ginsenoside Rb1; Ginsenoside 20(S)-Rg3

Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans by Hans Raj; Vinod Puthan Veetil; Wiktor Szymanski; Frank J. Dekker; Wim J. Quax; Ben L. Feringa; Dick B. Janssen; Gerrit J. Poelarends (pp. 385-397).

Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and pharmaceuticals. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. Ch-MAL is a dimer in solution, consisting of two identical subunits (∼49 kDa each), and requires Mg²⁺ and K⁺ ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 70°C (k _cat = 78 s⁻¹ and K _m = 16 mM). Heat inactivation assays showed that Ch-MAL is stable at 50°C for >4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate, and propylfumarate as substrates in the ammonia addition reaction. The enzyme also processes methylamine, ethylamine, hydrazine, hydroxylamine, and methoxylamine as nucleophiles that can replace ammonia in the addition to mesaconate, resulting in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess (>98% de). This newly identified thermostable MAL appears to be a potentially attractive biocatalyst for the stereoselective synthesis of aspartic acid derivatives on large (industrial) scale.

Keywords: Methylaspartate ammonia lyase; Thermostable enzyme; Carboxydothermus hydrogenoformans ; Enzyme catalysis; Amino acids

Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila by Evangelos Topakas; Maria Moukouli; Maria Dimarogona; Paul Christakopoulos (pp. 399-411).

A ferulic acid esterase (FAE) from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile), belonging to the carbohydrate esterase family 1 (CE-1), was functionally expressed in methylotrophic yeast Pichia pastoris. The putative FAE from the genomic DNA was successfully cloned in P. pastoris X-33 to confirm that the enzyme exhibits FAE activity. The recombinant FAE was purified to its homogeneity (39 kDa) and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme shows a preference for the hydrolysis of methyl caffeate and p-coumarate and a strong preference for the hydrolysis of n-butyl and iso-butyl ferulate. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose, whilst it was found capable of de-esterifying acetylated glucuronoxylans. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with an M3 xylanase from Trichoderma longibrachiatum (a maximum of 41% total FA released after 1 h incubation). Prediction of the secondary structure of MtFae1a was performed in the PSIPRED server whilst modelling the 3D structure was accomplished by the use of the HH 3D structure prediction server.

Keywords: Heterologous expression; Myceliophthora thermophila ; Pichia pastoris ; Ferulic acid esterase; Structural modelling

Characterization of a recombinant aryl β-glucosidase from Neosartorya fischeri NRRL181 by Dayanand Kalyani; Kyoung-Mi Lee; Manish Kumar Tiwari; Priyadharshini Ramachandran; Hoon Kim; In-Won Kim; Marimuthu Jeya; Jung-Kul Lee (pp. 413-423).

An isolated gene from Neosartorya fischeri NRRL181 encoding a β-glucosidase (BGL) was cloned, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,467 bp, capable of encoding a polypeptide of 488 amino acid residues. The gene was over-expressed in Escherichia coli, and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified recombinant BGL showed a high level of catalytic activity, with V _max of 886 μmol min⁻¹ mg-protein⁻¹ and a K _m of 68 mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature for enzyme activity was about 40°C, and the optimal pH was about 6.0. A homology model of N. fischeri BGL1 was constructed based on the X-ray crystal structure of Phanerochaete chrysosporium BGLA. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose shed light on the unique substrate specificity of N. fischeri BGL1 only towards pNPG.

Keywords: Characterization; β-Glucosidase; Glycoside hydrolase family 1; Homology modeling; Substrate specificity

Overexpression of the PHO84 gene causes heavy metal accumulation and induces Ire1p-dependent unfolded protein response in Saccharomyces cerevisiae cells by Augustin Minel Ofiteru; Lavinia Liliana Ruta; Codruta Rotaru; Ioana Dumitru; Cristian Dumitru Ene; Aurora Neagoe; Ileana Cornelia Farcasanu (pp. 425-435).

Pho84p, the protein responsible for the high-affinity uptake and transport of inorganic phosphate across the plasma membrane, is also involved in the low-affinity uptake of heavy metals in the Saccharomyces cerevisiae cells. In the present study, the effect of PHO84 overexpression upon the heavy metal accumulation by yeast cells was investigated. As PHO84 overexpression triggered the Ire1p-dependent unfolded protein response, abundant plasma membrane Pho84p could be achieved only in ire1Δ cells. Under environmental surplus, PHO84 overexpression augmented the metal accumulation by the wild type, accumulation that was exacerbated by the IRE1 deletion. The pmr1Δ cells, lacking the gene that encodes the P-type ATPase ion pump that transports Ca²⁺ and Mn²⁺ into the Golgi, hyperaccumulated Mn²⁺ even from normal medium when overexpressing PHO84, a phenotype which is rather restricted to metal-hyperaccumulating plants.

Keywords: PHO84 ; Overexpression; Heavy metal accumulation; Saccharomyces cerevisiae

Genes involved in alkane degradation in the Alcanivorax hongdengensis strain A-11-3 by Wanpeng Wang; Zongze Shao (pp. 437-448).

Alcanivorax hongdengensis A-11-3 is a newly identified type strain isolated from the surface water of the Malacca and Singapore Straits that can degrade a wide range of alkanes. To understand the degradation mechanism of this strain, the genes encoding alkane hydroxylases were obtained by PCR screening and shotgun sequencing of a genomic fosmid library. Six genes involved in alkane degradation were found, including alkB1, alkB2, p450-1, p450-2, p450-3 and almA. Heterogeneous expression analysis confirmed their functions as alkane oxidases in Pseudomonas putida GPo12 (pGEc47ΔB) or Pseudomonas fluorescens KOB2Δ1. Q-PCR revealed that the transcription of alkB1 and alkB2 was enhanced in the presence of n-alkanes C₁₂ to C₂₄; three p450 genes were up-regulated by C₈–C₁₆ n-alkanes at different levels, whereas enhanced expression of almA was observed when strain A-11-3 grew with long-chain alkanes (C₂₄ to C₃₆). In the case of branched alkanes, pristane significantly enhanced the expression of alkB1, p450-3 and almA. The six genes enable strain A-11-3 to degrade short (C₈) to long (C₃₆) alkanes that are straight or branched. The ability of A. hongdengensis A-11-3 to thrive in oil-polluted marine environments may be due to this strain’s multiple systems for alkane degradation and its range of substrates.

Keywords: Marine oil pollution; Biodegradation; Alkane hydroxylase; Gene cluster; Alcanivorax hongdengensis

Engineering Corynebacterium glutamicum for the production of pyruvate by Stefan Wieschalka; Bastian Blombach; Bernhard J. Eikmanns (pp. 449-459).

A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM l-valine, 28 mM l-alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicum △aceE △pqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD⁺-dependent l-lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (△C–T IlvN). The latter modification abolished overflow metabolism towards l-valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN produced about 190 mM pyruvate with a Y _P/S of 1.36 mol per mol of glucose; however, it still secreted significant amounts of l-alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced l-alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0–5% dissolved oxygen), the newly constructed strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN △alaT △avtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g _(CDW) ⁻¹ h⁻¹ (i.e., 0.08 g g_(CDW) ⁻¹ h⁻¹) in the production phase.

Keywords: Corynebacterium glutamicum ; Pyruvate production; Pyruvate dehydrogenase complex; Pyruvate:quinone oxidoreductase; NAD⁺-dependent l-lactate dehydrogenase; Acetohydroxyacid synthase; Transaminases; Industrial biotechnology

Isolation, characterization, and quantification of Clostridium kluyveri from the bovine rumen by Paul J. Weimer; David M. Stevenson (pp. 461-466).

A strain of Clostridium kluyveri was isolated from the bovine rumen in a medium containing ethanol as an electron donor and acetate and succinate (common products of rumen fermentation) as electron acceptors. The isolate displayed a narrow substrate range but wide temperature and pH ranges atypical of ruminal bacteria and a maximum specific growth rate near the typical liquid dilution rate of the rumen. Quantitative real-time PCR revealed that C. kluyveri was widespread among bovine ruminal samples but was present at only very low levels (0.00002% to 0.0002% of bacterial 16S rRNA gene copy number). However, the species was present in much higher levels (0.26% of bacterial 16S rRNA gene copy number) in lucerne silage (but not maize silage) that comprised much of the cows’ diet. While C. kluyveri may account for several observations regarding ethanol utilization and volatile fatty acid production in the rumen, its population size and growth characteristics suggest that it is not a significant contributor to ruminal metabolism in typical dairy cattle, although it may be a significant contributor to silage fermentation. The ability of unadapted cultures to produce substantial levels (12.8 g L⁻¹) of caproic (hexanoic) acid in vitro suggests that this strain may have potential for industrial production of caproic acid.

Keywords: Butyric acid; Caproic acid; Ethanol; Clostridium ; Rumen; Silage

Synthesis and characterization of the antibacterial potential of ZnO nanoparticles against extended-spectrum β-lactamases-producing Escherichia coli and Klebsiella pneumoniae isolated from a tertiary care hospital of North India by Mohammad Azam Ansari; Haris M. Khan; Aijaz A. Khan; Asfia Sultan; Ameer Azam (pp. 467-477).

The reemergence of infectious diseases and the continuous development of multidrug resistance among a variety of disease-causing bacteria in clinical setting pose a serious threat to public health worldwide. Extended-spectrum β-lactamases (ESBLs) that mediate resistance to third-generation cephalosporin are now observed all over the world in all species of Enterobacteriaceae, especially Escherichia coli and Klebsiella pneumoniae. In this work, ZnO nanoparticles (NPs) were synthesized by the sol–gel method and characterized by powder X-ray diffraction, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The image of synthesized ZnO NPs appeared spherical in SEM with a diameter of ≈19 nm and as hexagonal crystal in AFM. Clinical isolates were assessed for ESBL production and shown to be sensitive to ZnO NPs by different methods such as minimal inhibitory concentration (MIC) and minimal bactericidal concentration, time-dependent growth inhibition assay, well diffusion agar methods and estimation of colony forming units (CFU) of bacteria. The lowest MIC value for E. coli and K. pneumoniae was found to be 500 μg/ml. The results showed that ZnO NPs at 1,000 μg/ml completely inhibit the bacterial growth. The antibacterial effect of ZnO nanoparticles was gradual, but time- and concentration-dependent. The maximum inhibition zone at100 μg/ml for E. coli and K. pneumoniae was 22 and 20 mm, respectively. With the increasing ZnO NP loading, there is significant reduction in the numbers of CFU. At the concentration of 1,000 μg/ml, the decline in per cent survival of E. coli and K. pneumoniae was found to be 99.3% and 98.6%, respectively.

Keywords: ZnO nanoparticles; ESBL; E. coli ; K. pneumoniae ; AFM; CFU

Insulin-producing cells from human adipose tissue-derived mesenchymal stem cells detected by atomic force microscope by Qiping Shi; Simin Luo; Hua Jin; Jiye Cai; Haiying Jia; Lie Feng; Xiaohua Lu (pp. 479-486).

We successfully differentiated human adipose tissue-derived mesenchymal stem cells (haMSCs) into insulin-producing cells (IPCs) in vitro and did not use any insulin which might be absorbed by cells during in vitro culture. Expression of insulin gene was massively increased by 28,000-fold at day 12 compared with haMSCs (P < 0.05). IPCs could secrete insulin after glucose was stimulated. The higher the concentration of glucose, the more production of insulin was noted. We reported AFM images of IPCs for the first time. AFM images showed that the sizes of cells were similar to each other, and all IPC surface had a porous structure in the cytoplasm area. In sugar-free group, the size of holes was similar (diameter, 1,086.98 ± 156.70 nm; depth, 185.22 ± 52.14 nm). In higher sugar-stimulated group, there were more holes with bigger diameter and smaller depth. (diameter, 3,183.65 ± 2,229.18 nm; depth 109.42 ± 56.26 nm, P < 0.05). We found that the hole diameter and depth could change with the concentration of glucose in media. Concurrently, laser scanning confocal microscopy images indicated that cortical actin network beneath plasma membrane in IPCs was dense and continuous. After glucose stimulation, we found the actin web depolymerized and became discontinuous in IPCs. We speculated that diameter augmentation of holes located in the cytoplasm area in IPCs was one manifestation of excytosis increase.

Keywords: Human adipose tissue-derived mesenchymal stem cells; Insulin-producing cells; F-actin; Atomic force microscopy

An efficient transformation method for Bacillus subtilis DB104 by Ljubica Vojcic; Dragana Despotovic; Ronny Martinez; Karl-Heinz Maurer; Ulrich Schwaneberg (pp. 487-493).

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10³ transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 10⁵ transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.

Keywords: B. subtilis DB104; Directed evolution; Natural competence; Transformation protocol

Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice by Tao Tang; Lianfeng Zhang; Ran Gao; Yunping Dai; Fanchao Meng; Ying Li (pp. 495-503).

Bacterial magnetic particles (BMPs) are of interest as potential carriers of bioactive macromolecules, drugs, or liposomes. In this study, a high-pressure homogenizer was used to disrupt Magnetospirillum gryphiswaldense strain MSR-1 cells, and BMPs were purified. BMPs were labeled with fluorescence reagent 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocianin perchlorate (DiI) and injected into the tail vein of BALB/c nude mice. Distribution of fluorescence signals of DiI–BMPs in vivo was examined using a whole-body fluorescence imaging system. The result showed that fluorescence signals were detected in liver, stomach, intestine, lungs, and spleen. However, transmission electron microscopy of ultrathin sections indicated that BMPs were mainly present in liver and lungs, but not in the other organs. BMPs could be useful as carriers for targeted drug therapy of diseases of the liver or lung.

Keywords: Bacterial magnetic particles; Labeling; Fluorescence imaging; Nude mice; Tracking

Bioremediation of β-cypermethrin and 3-phenoxybenzaldehyde contaminated soils using Streptomyces aureus HP-S-01 by Shaohua Chen; Peng Geng; Ying Xiao; Meiying Hu (pp. 505-515).

Using laboratory and field experiments, the ability of Streptomyces aureus HP-S-01 to eliminate β-cypermethrin (β-CP) and its metabolite 3-phenoxybenzaldehyde (3-PBA) in soils was investigated. In the laboratory, 80.5% and 73.1% of the initial dose of β-CP and 3-PBA (50 mg kg⁻¹) was removed in sterilized soils within 10 days, respectively, while in the same period, disappearance rate of β-CP and 3-PBA in non-sterilized soils was higher and reached 87.8% and 79.3%, respectively. Furthermore, the disappearance process followed the first-order kinetics and the half-life (T _1/2) for β-CP and 3-PBA reduced by 20.3–52.9 and 133.7–186.8 days, respectively, as compared to the controls. The addition of sucrose to the soils enhanced the ability of strain HP-S-01 to eliminate β-CP and 3-PBA. Similar results were observed in the field experiments. The introduced strain HP-S-01 quickly adapted to the environment and rapidly removed β-CP and 3-PBA without any lag phases in the field experiments. Compared with the controls, 47.9% and 67.0% of applied dose of β-CP and 3-PBA was removed from the soils without extra carbon sources and 52.5% and 73.3% of β-CP and 3-PBA was eliminated in soils supplemented with sucrose within 10 days, respectively. Analysis of β-CP degradation products in soil indicated that the tested strain transform β-CP to 3-PBA and α-hydroxy-3-phenoxy-benzeneacetonitrile. However, both intermediates were transient and they disappeared after 10 days. Therefore, the selected actinomyces strain HP-S-01 is suitable for the efficient and rapid bioremediation of β-CP contaminated soils.

Keywords: β-Cypermethrin; 3-Phenoxybenzaldehyde; Bioremediation; Streptomyces aureus HP-S-01; Kinetics; Soil

Achieving nitrite accumulation in a continuous system treating low-strength domestic wastewater: switchover from batch start-up to continuous operation with process control by Yongzhen Peng; Jianhua Guo; Harald Horn; Xiong Yang; Shuying Wang (pp. 517-526).

Although biological nitrogen removal via nitrite is recognized as one of the cost-effective and sustainable biological nitrogen removal processes, nitrite accumulation has proven difficult to achieve in continuous processes treating low-strength nitrogenous wastewater. Partial nitrification to nitrite was achieved and maintained in a lab-scale completely stirred tank reactor (CSTR) treating real domestic wastewater. During the start-up period, sludge with ammonia-oxidizing bacteria (AOB) but no nitrite-oxidizing bacteria (NOB) was obtained by batch operation with aeration time control. The nitrifying sludge with the dominance of AOB was then directly switched into continuous operation. It was demonstrated that partial nitrification to nitrite in the continuous system could be repeatedly and reliably achieved using this start-up strategy. The ratio of dissolved oxygen to ammonium loading rate (DO/ALR) was critical to maintain high ammonium removal efficiency and nitrite accumulation ratio. Over 85% of nitrite accumulation ratio and more than 95% of ammonium removal efficiency were achieved at DO/ALR ratios in an optimal range of 4.0–6.0 mg O₂/g N d, even under the disturbances of ammonium loading rate. Microbial population shift was investigated, and fluorescence in situ hybridization analysis indicated that AOB were the dominant nitrifying bacteria over NOB when stable partial nitrification was established.

Keywords: Biological nitrogen removal; Partial nitrification to nitrite; Process control; Ammonia-oxidizing bacteria; Sludge population optimization; Biological wastewater treatment

Internal loop photobiodegradation reactor (ILPBR) for accelerated degradation of sulfamethoxazole (SMX) by Ning Yan; Siqing Xia; Linke Xu; Jun Zhu; Yongming Zhang; Bruce E. Rittmann (pp. 527-535).

The internal loop photobiodegradation reactor (ILPBR) was evaluated for the degradation of the pharmaceutical sulfamethoxazole (SMX) using batch experiments following three protocols: photolysis alone (P), biodegradation alone (B), and intimately coupled photolysis and biodegradation (P&B). SMX was removed more rapidly by P&B than by either P or B alone, and the corresponding dissolved organic carbon (DOC) removals by P&B also were higher. The faster SMX removal probably was due to a synergy between photolysis and the rapid biodegradation of SMX by the biofilm. The greater DOC removal was brought about by the presence of biofilm bacteria able to biodegrade photolysis products. Ammonium N released during photolysis of SMX gave more evidence for the formation of intermediates and was enough in P&B experiments to support bioactivity when no other N was supplied. Clone libraries performed on the biofilms before and after the P&B experiments showed profound changes in the microbial community. Whereas Rhodopirellula baltica and Methylibium petroleiphilum PM1 dominated the biofilm after the B experiments, they were replaced by Micrococcus luteus, Delftia acidovorans, and Oligotropha carboxidovorans after the P&B experiments. The changes in microbial community structure mirrored the change in function in the P&B experiments: SMX biodegradation (presumably the roles of R. baltica and M. petroleiphilum) was out-competed by SMX photolysis, but biodegradation of photolysis products (most likely by M. luteus and D. acidovorans) became important. The higher removal rates of SMX and DOC, as well as the changes in microbial community structure, confirm the value of intimately coupling photolysis with biodegradation in the ILPBR.

Keywords: Biodegradation; Biofilm; Microbial community structure; Photolysis; Sulfamethoxazole

Microbial community structure elucidates performance of Glyceria maxima plant microbial fuel cell by Ruud A. Timmers; Michael Rothballer; David P. B. T. B. Strik; Marion Engel; Stephan Schulz; Michael Schloter; Anton Hartmann; Bert Hamelers; Cees Buisman (pp. 537-548).

The plant microbial fuel cell (PMFC) is a technology in which living plant roots provide electron donor, via rhizodeposition, to a mixed microbial community to generate electricity in a microbial fuel cell. Analysis and localisation of the microbial community is necessary for gaining insight into the competition for electron donor in a PMFC. This paper characterises the anode–rhizosphere bacterial community of a Glyceria maxima (reed mannagrass) PMFC. Electrochemically active bacteria (EAB) were located on the root surfaces, but they were more abundant colonising the graphite granular electrode. Anaerobic cellulolytic bacteria dominated the area where most of the EAB were found, indicating that the current was probably generated via the hydrolysis of cellulose. Due to the presence of oxygen and nitrate, short-chain fatty acid-utilising denitrifiers were the major competitors for the electron donor. Acetate-utilising methanogens played a minor role in the competition for electron donor, probably due to the availability of graphite granules as electron acceptors.

Keywords: 454 amplicon sequencing; Geobacter ; Microbial community; Plant microbial fuel cell; Renewable energy; Rhizosphere

Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris by Pavel Přibyl; Vladislav Cepák; Vilém Zachleder (pp. 549-561).

We tested 10 different Chlorella and Parachlorella strains under lipid induction growth conditions in autotrophic laboratory cultures. Between tested strains, substantial differences in both biomass and lipid productivity as well as in the final content of lipids were found. The most productive strain (Chlorella vulgaris CCALA 256) was subsequently studied in detail. The availability of nitrates and/or phosphates strongly influenced growth and accumulation of lipids in cells by affecting cell division. Nutrient limitation substantially enhanced lipid productivity up to a maximal value of 1.5 g l⁻¹ day⁻¹. We also demonstrated the production of lipids through large-scale cultivation of C. vulgaris in a thin layer photobioreactor, even under suboptimal conditions. After 8 days of cultivation, maximal lipid productivity was 0.33 g l⁻¹ day⁻¹, biomass density was 5.7 g l⁻¹ dry weight and total lipid content was more than 30% dry weight. C. vulgaris lipids comprise fatty acids with a relatively high degree of saturation compared with canola oil offering a possible alternative to the use of higher plant oils.

Keywords: Chlorella ; Cultivation; Fatty acids; Lipid; Oil; Parachlorella ; Photobioreactor; Productivity

Erratum to: Insight into the roles of hypoxanthine and thymidine on cultivating antibody-producing CHO cells: cell growth, antibody production and long-term stability by Fei Chen; Li Fan; Jiaqi Wang; Yan Zhou; Zhaoyang Ye; Liang Zhao; Wen-Song Tan (pp. 563-563).

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Applied Microbiology and Biotechnology (v.94, #2)

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Applied Microbiology and Biotechnology (v.94, #2)