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Applied Microbiology and Biotechnology (v.89, #3)

Biodiversity, bioactive natural products and biotechnological potential of plant-associated endophytic actinobacteria by Sheng Qin; Ke Xing; Ji-Hong Jiang; Li-Hua Xu; Wen-Jun Li (pp. 457-473).

Endophytic actinobacteria, which exist in the inner tissues of living plants, have attracted increasing attention among taxonomists, ecologists, agronomists, chemists and evolutionary biologists. Numerous studies have indicated that these prolific actinobacteria appear to have a capacity to produce an impressive array of secondary metabolites exhibiting a wide variety of biological activity, such as antibiotics, antitumor and anti-infection agents, plant growth promoters and enzymes, and may contribute to their host plants by promoting growth and enhancing their ability of withstanding the environmental stresses. These microorganisms may represent an underexplored reservoir of novel species of potential interest in the discovery of novel lead compounds and for exploitation in pharmaceutical, agriculture and industry. This review focuses on new findings in the isolation methods, bio- and chemical diversity of endophytic actinobacteria and reveals the potential biotechnological application. The facing problems and strategies for biodiversity research and bioactive natural products producing are also discussed.

Keywords: Endophytic actinobacteria; Biodiversity; Natural products; Biotechnology

Antimicrobial polymers: mechanism of action, factors of activity, and applications by Larisa Timofeeva; Natalia Kleshcheva (pp. 475-492).

Complex epidemiological situation, nosocomial infections, microbial contamination, and infection risks in hospital and dental equipment have led to an ever-growing need for prevention of microbial infection in these various areas. Macromolecular systems, due to their properties, allow one to efficiently use them in various fields, including the creation of polymers with the antimicrobial activity. In the past decade, the intensive development of a large class of antimicrobial macromolecular systems, polymers, and copolymers, either quaternized or functionalized with bioactive groups, has been continued, and they have been successfully used as biocides. Various permanent microbicidal surfaces with non-leaching polymer antimicrobial coatings have been designed. Along with these trends, new moderately hydrophobic polymer structures have been synthesized and studied, which contain protonated primary or secondary/tertiary amine groups that exhibited rather high antimicrobial activity, often unlike their quaternary analogues. This mini-review briefly highlights and summarizes the results of studies during the past decade and especially in recent years, which concern the mechanism of action of different antimicrobial polymers and non-leaching microbicidal surfaces, and factors influencing their activity and toxicity, as well as major applications of antimicrobial polymers.

Keywords: Bacteria; Antimicrobial activity; Quaternary/non-quaternary polymers; Mechanism of action

Ionic liquids for two-phase systems and their application for purification, extraction and biocatalysis by Sebastian Oppermann; Florian Stein; Udo Kragl (pp. 493-499).

The development of biotechnological processes using novel two-phase systems based on molten salts known as ionic liquids (ILs) got into the focus of interest. Many new approaches for the beneficial application of the interesting solvent have been published over the last years. ILs bring beneficial properties compared to organic solvents like nonflammability and nonvolatility. There are two possible ways to use the ILs: first, the hydrophobic ones as a substitute for organic solvents in pure two-phase systems with water and second, the hydrophilic ones in aqueous two-phase systems (ATPS). To effectively utilise IL-based two-phase systems or IL-based ATPS in biotechnology, extensive experimental work is required to gain the optimal system parameters to ensure selective extraction of the product of interest. This review will focus on the most actual findings dealing with the basic driving forces for the target extraction in IL-based ATPS as well as presenting some selected examples for the beneficial application of ILs as a substitute for organic solvents. Besides the research focusing on IL-based two-phase systems, the “green aspect” of ILs, due to their negligible vapour pressure, is widely discussed. We will present the newest results concerning ecotoxicity of ILs to get an overview of the state of the art concerning ILs and their utilisation in novel two-phase systems in biotechnology.

Keywords: Ionic liquids; Biotechnology; Extraction; Two-phase systems; Downstream processing

RNAi as a potential tool for biotechnological applications in fungi by Tomer M. Salame; Carmit Ziv; Yitzhak Hadar; Oded Yarden (pp. 501-512).

RNA interference (RNAi) is a post-transcriptional gene-silencing (PTGS) phenomenon in which double-stranded RNA (dsRNA) triggers the degradation of homologous mRNA species, thereby reducing gene expression. In fungi, the use of RNAi as a tool for reverse genetics, aimed at modification of gene expression, is constantly growing, with more than 40 species already proven to be responsive. This technology has the ability to co-down-regulate the expression of several genes; however, this trait also makes it susceptible to non-target effects, which can be addressed using both available and developing bioinformatic tools. Moreover, the functionality of absorbed exogenous RNAi molecules, and the various classes of small RNAs found in fungi, offer great versatility and flexibility in acquiring the desired effects on gene expression, even without the necessity to genetically modify the targeted strain. There is an emerging role for RNAi as a potential tool for biotechnological applications. This is evident from current investigations in fungi, demonstrating the contribution of RNAi to progress research and applications in biomaterials production, bioconversion, plant fungal interactions and virulence factors of human pathogens. Possible problems and prospects for the use of RNAi in fungal biotechnology are discussed.

Keywords: RNA interference (RNAi); RNA silencing

A review—biosynthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate ester: recent advances and future perspectives by Qi Ye; Pingkai Ouyang; Hanjie Ying (pp. 513-522).

Ethyl (S)-4-chloro-3-hydroxybutanoate ester ((S)-CHBE) is a precursor of enantiopure intermediates used for the production of chiral drugs, including the cholesterol-lowering 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitors (statins). The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate ester (COBE) to (S)-CHBE by biocatalysis has several positive attributes, including low cost, mild reaction conditions, high yield, and a high level of enantioselectivity. During genome database mining of the yeast Pichia stipitis, our group found two novel carbonyl reductases (PsCRI and PsCRII) that have a promising future for the industrial production of (S)-CHBE with >99% enantiomeric excess. This review covers the main process of biosynthesis of (S)-CHBE: screening of microorganisms that catalyze the reduction of COBE to (S)-CHBE (I); gene cloning, expression, and characterization of carbonyl reductases for the production of (S)-CHBE in Escherichia coli (II); development of cofactor generation systems for regenerating cofactors (III); and biocatalysis of COBE to (S)-CHBE by recombinant E. coli (IV).

Keywords: Ethyl 4-chloro-3-oxobutanoate ester; Ethyl (S)-4-chloro-3-hydroxybutanoate ester; Carbonyl reductase; Biocatalysis; Stereoselective reduction

Bioavailability of organic compounds solubilized in nonionic surfactant micelles by Zhilong Wang (pp. 523-534).

Whether direct availability of organic compound solubilized in nonionic surfactant micelles (bioavailability) in a bioremediation or biotransformation process is uncertain to some extent, which is partially attributed to the difficulty by direct experimental determination. In another point of view, it should be ascribed to the fuzzy concept about the solubilization of organic compound in a nonionic surfactant micelle aqueous solution. In this mini-review, the solubilization of organic compound in surfactant micelles aqueous solution is fully discussed; especially saturated solubilization and unsaturated solubilization have been emphasized. Then the current methods for estimation of bioavailability of organic compounds solubilized in micelles are introduced, in which the possible drawbacks of each method are stressed. Finally, the conclusion that organic compound solubilized in micelles is unavailable directly by microbes has been drawn and the intensification of bioremediation or biotransformation by nonionic surfactant micelle aqueous solution is contributed to enhancement of the hydrophobic organic compounds dissolution.

Keywords: Bioavailability; Solubilization; Bioremediation; Nonionic surfactant; Micelles

Biochemistry of lactone formation in yeast and fungi and its utilisation for the production of flavour and fragrance compounds by Cynthia Romero-Guido; Isabel Belo; Thi Minh Ngoc Ta; Lan Cao-Hoang; Mohamed Alchihab; Nelma Gomes; Philippe Thonart; Jose A. Teixeira; Jacqueline Destain; Yves Waché (pp. 535-547).

The consumers’ demand for natural flavour and fragrances rises. To be natural, compounds have to result from the extraction of natural materials and/or to be transformed by natural means such as the use of enzymes or whole cells. Fungi are able to transform some fatty acids into lactones that can thus be natural. Although some parts of this subject have been reviewed several times, the present article proposes to review the different pathways utilised, the metabolic engineering strategies and some current concerns on the reactor application of the transformation including scaling up data. The main enzymatic steps are hydroxylation and β-oxidation in the traditional way, and lactone desaturation or Baeyer–Villiger oxidation. Although the pathway to produce γ-decalactone is rather well known, metabolic engineering strategies may result in significant improvements in the productivity. For the production of other lactones, a key step is the hydroxylation of fatty acids. Beside the biotransformation, increasing the production of the various lactones requires from biotechnologists to solve two main problems which are the toxicity of lactones toward the producing cell and the aeration of the emulsified reactor as the biochemical pathway is very sensitive to the level of available oxygen. The strategies employed to resolve these problems will be presented.

Keywords: Lactone; Yeast; Fungi; Yarrowia ; Rhodotorula ; Aroma; Lipids; Metabolism; Hydroxylation; β-oxidation; Oxygen transfer; Bioreactor; Up scaling; Emulsion; Cell wall; Membrane; Fluidity; Toxicity; Trapping; Immobilisation

The unconventional antimicrobial peptides of the classical propionibacteria by Therese Faye; Helge Holo; Thor Langsrud; Ingolf F. Nes; Dag A. Brede (pp. 549-554).

The classical propionibacteria produce genetically unique antimicrobial peptides, whose biological activities are without equivalents, and to which there are no homologous sequences in public databases. In this review, we summarize the genetics, biochemistry, biosynthesis, and biological activities of three extensively studied antimicrobial peptides from propionibacteria. The propionicin T1 peptide constitutes a bona fide example of an unmodified general secretory pathway (sec)-dependent bacteriocin, which is bactericidal towards all tested species of propionibacteria except Propionibacterium freudenreichii. The PAMP antimicrobial peptide represents a novel concept within bacterial antagonism, where an inactive precursor protein is secreted in large amounts, and which activation appears to rely on subsequent processing by proteases in its resident milieu. Propionicin F is a negatively charged bacteriocin that displays an intraspecies bactericidal inhibition spectrum. The biosynthesis of propionicin F appears to proceed through a series of unusual events requiring both N- and C-terminal processing of a precursor protein, which probably requires the radical SAM superfamily enzyme PcfB.

Keywords: Propionibacterium; Antimicrobial peptides; Genetics; Biochemistry

Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces dendrorhous by Isabell Schmidt; Hendrik Schewe; Sören Gassel; Chao Jin; John Buckingham; Markus Hümbelin; Gerhard Sandmann; Jens Schrader (pp. 555-571).

The oxygenated β-carotene derivative astaxanthin exhibits outstanding colouring, antioxidative and health-promoting properties and is mainly found in the marine environment. To satisfy the growing demand for this ketocarotenoid in the feed, food and cosmetics industries, there are strong efforts to develop economically viable bioprocesses alternative to the current chemical synthesis. However, up to now, natural astaxanthin from Haematococcus pluvialis, Phaffia rhodozyma or Paracoccus carotinifaciens has not been cost competitive with chemically synthesized astaxanthin, thus only serving niche applications. This review illuminates recent advances made in elucidating astaxanthin biosynthesis in P. rhodozyma. It intensely focuses on strategies to increase astaxanthin titers in the heterobasidiomycetous yeast by genetic engineering of the astaxanthin pathway, random mutagenesis and optimization of fermentation processes. This review emphasizes the potential of P. rhodozyma for the biotechnological production of astaxanthin in comparison to other natural sources such as the microalga H. pluvialis, other fungi and transgenic plants and to chemical synthesis.

Keywords: Astaxanthin; Astaxanthin biosynthesis; Astaxanthin synthase; Bioprocess engineering; Metabolic engineering; Phaffia rhodozyma

Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols by Chao Yu; Yujin Cao; Huibin Zou; Mo Xian (pp. 573-583).

Confronted with the gradual and inescapable exhaustion of the earth’s fossil energy resources, the bio-based process to produce platform chemicals from renewable carbohydrates is attracting growing interest. Escherichia coli has been chosen as a workhouse for the production of many valuable chemicals due to its clear genetic background, convenient to be genetically modified and good growth properties with low nutrient requirements. Rational strain development of E. coli achieved by metabolic engineering strategies has provided new processes for efficiently biotechnological production of various high-value chemical building blocks. Compared to previous reviews, this review focuses on recent advances in metabolic engineering of the industrial model bacteria E. coli that lead to efficient recombinant biocatalysts for the production of high-value organic acids like succinic acid, lactic acid, 3-hydroxypropanoic acid and glucaric acid as well as alcohols like 1,3-propanediol, xylitol, mannitol, and glycerol with the discussion of the future research in this area. Besides, this review also discusses several platform chemicals, including fumaric acid, aspartic acid, glutamic acid, sorbitol, itaconic acid, and 2,5-furan dicarboxylic acid, which have not been produced by E. coli until now.

Keywords: Escherichia coli ; Metabolic pathway; Organic acid; Alcohol; Platform chemicals

Evaluation of rhamnolipid production capacity of Pseudomonas aeruginosa PAO1 in comparison to the rhamnolipid over-producer strains DSM 7108 and DSM 2874 by Markus Michael Müller; Barbara Hörmann; Michaela Kugel; Christoph Syldatk; Rudolf Hausmann (pp. 585-592).

A lack of understanding of the quantitative rhamnolipid production regulation in bioreactor cultivations of Pseudomonas aeruginosa and the absence of respective comparative studies are important reasons for achieving insufficient productivities for an economic production of these biosurfactants. The Pseudomonas strains DSM 7108 and DSM 2874 are described to be good rhamnolipid over-producers. The strain PAO1 on the other hand is the best analyzed type strain for genetic regulation mechanisms in the species P. aeruginosa. These three strains were cultivated in a 30-L bioreactor with a medium containing nitrate and sunflower oil as sole C-source at 30 and 37 °C. The achieved maximum rhamnolipid concentrations varied from 7 to 38 g/L, the volumetric productivities from 0.16 to 0.43 g/(L·h), and the cellular yield from 0.67 to 3.15 g/g, with PAO1 showing the highest results for all of these variables. The molar di- to mono-rhamnolipid ratio changed during the cultivations; it was strain dependent but not significantly influenced by the temperature. This study explicitly shows that the specific rhamnolipid synthesis rate per cell follows secondary metabolite-like courses coinciding with the transition to the stationary phase of typical logistic growth behavior. However, the rhamnolipid synthesis was already induced before N-limitation occurred.

Keywords: Rhamnolipid; Biosurfactant; Renewable resources; Pseudomonas ; Specific productivity; Glycolipid

A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium by Jens Kroll; Stefan Klinter; Alexander Steinbüchel (pp. 593-604).

Hitherto the production of the biopolymer cyanophycin (CGP) using recombinant Escherichia coli strains and cheap mineral salts medium yielded only trace amounts of CGP (<0.5%, w/w) of the cell dry matter (CDM). This was probably due to the instability of the plasmids encoding the cyanophycin synthetase.In this study, we developed an anabolism-based media-dependent plasmid addiction system (PAS) to enhance plasmid stability, and we established a process based on a modified mineral salts medium yielding a CGP content of 42% (w/w) at the maximum without the addition of amino acids to the medium for the first time. This PAS is based on different lysine biosynthesis pathways and consists of two components: (1) a knockout of the chromosomal dapE disrupts the native succinylase pathway in E. coli and (2) the complementation by the plasmid-encoded artificial aminotransferase pathway mediated by the dapL gene from Synechocystis sp. PCC 6308, which allows the synthesis of the essential lysine precursor L,L-2,6-diaminopimelate. In addition, this plasmid also harbors cphAC595S, an engineered cyanophycin synthetase gene responsible for CGP production.Cultivation experiments in Erlenmeyer flask and also in bioreactors in mineral salts medium without antibiotics revealed an at least 4.5-fold enhanced production of CGP in comparison to control cultivations without PAS.Fermentation experiments with culture volume of up to 400 l yielded a maximum of 18% CGP (w/w) and a final cell density of 15.2 g CDM/l. Lactose was used constantly as an effective inducer and carbon source. Thus, we present a convenient option to produce CGP with E. coli at a technical scale without the need to add antibiotics or amino acids using the mineral salts medium designed in this study.

Keywords: Cyanophycin; DapE; DapL; Mineral salts medium; Plasmid addiction system; Succinylase pathway

Microbial community differences between propionate-fed microbial fuel cell systems under open and closed circuit conditions by Daniel Aguirre de Cárcer; Phuc Thi Ha; Jae Kyung Jang; In Seop Chang (pp. 605-612).

We report the electrochemical characterization and microbial community analysis of closed circuit microbial fuel cells (CC-MFCs) and open circuit (OC) cells continuously fed with propionate as substrate. Differences in power output between MFCs correlated with their polarization behavior, which is related to the maturation of the anodophilic communities. The microbial communities residing in the biofilm growing on the electrode, biofouled cation-exchange membrane and anodic chamber liquor of OC-and CC-MFCs were characterized by restriction fragment length polymorphism screening of 16S rRNA gene clone libraries. The results show that the CC-MFC anode was enriched in several microorganisms related to known electrochemically active and dissimilatory Fe(III) reducing bacteria, mostly from the Geobacter spp., to the detriment of Bacteroidetes abundant in the OC-MFC anode. The results also evidenced the lack of a specific pelagic community in the liquor sample. The biofilm growing on the cation-exchange membrane of the CC-MFC was found to be composed of a low-diversity community dominated by two microaerophilic species of the Achromobacter and Azovibrio genus.

Keywords: Microbial fuel cell; Propionate; Microbial community; Geobacter

Optimization of cold-active chitinase production from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 by Se Jong Han; Heeyong Park; Sung Gu Lee; Hong Kum Lee; Joung Han Yim (pp. 613-621).

In the present study, cultivation conditions and medium components were optimized using statistical design and analysis to enhance the production of Chi21702, a cold-active extracellular chitinase from the Antarctic bacterium Sanguibacter antarcticus KOPRI 21702. Identification of significant carbon sources and other key elements was performed using a statistical design technique. Chitin and glycerol were selected as main carbon sources, and the ratio of complex nitrogen sources to carbon sources was determined to be 0.5. Among 15 mineral components included in basal medium, NaCl, Fe(C₆H₅O₇), and MgCl₂ were found to have the most influence on Chi21702 production. The optimal parameters of temperature, initial pH, and dissolved oxygen level were found to be 25°C, 6.5, and above 30% of air saturation, respectively. The maximum Chi21702 activity obtained under the optimized conditions was 90 U/L. Through statistical optimization methods, a 7.5-fold increase in Chi21702 production was achieved over unoptimized conditions. Chi21702 showed relatively high activity, even at low temperatures close to 0°C. The information obtained in the present study could be applied to the production of cold-active endochitinase on a large scale, suitable for a process at low temperature in industry.

Keywords: Antarctic bacterium; Cold-active; Endochitinase; Sanguibacter antarcticus ; Statistical optimization

Elaboration of antibiofilm materials by chemical grafting of an antimicrobial peptide by Jean-Fabrice Yala; Pascal Thebault; Arnaud Héquet; Vincent Humblot; Claire-Marie Pradier; Jean-Marc Berjeaud (pp. 623-634).

A peptide antibiotic, gramicidin A, was covalently bound to cystamine self-assembled monolayers on gold surfaces. Each step of the surface functionalization was characterized by polarization modulation infrared reflection absorption spectroscopy and X-ray photoelectron spectroscopy. The antimicrobial activity of the anchored gramicidin was tested against three Gram-positive bacteria (Listeria ivanovii, Enterococcus faecalis, and Staphylococcus aureus), the Gram-negative bacterium Escherichia coli and the yeast Candida albicans. The results revealed that the adsorbed gramicidin reduced, from 60% for E. coli to 90% for C. albicans, the number of culturable microorganisms attached to the surface. The activity was proven to be persistent overtime, up to 6 months after the first use. The bacteria attached to the functionalized surfaces were permeabilized as shown by confocal microscopy. Taken together, these results indicate a bacteriostatic mode of action of the immobilized peptide. Finally, using green fluorescent protein-expressing bacteria, it was shown that the development of a bacterial biofilm was delayed on peptide-grafted surfaces for at least 24 h.

Keywords: Antimicrobial surfaces; Gramicidin; Biofilm

Cloning and characterization of a rhamnose isomerase from Bacillus halodurans by Ponnandy Prabhu; Thanh Thi Ngoc Doan; Marimuthu Jeya; Lin-Woo Kang; Jung-Kul Lee (pp. 635-644).

Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an l-rhamnose isomerase (l-RhI). The identified l -RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k _cat of 8,971 min⁻¹ and a k _cat/K _m of 17 min⁻¹ mM⁻¹ for l-rhamnose. Although l-RhIs have been characterized from several other sources, B. halodurans l-RhI is distinguished from other l-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans l-RhI a good choice for industrial applications. This work describes one of the most thermostable l-RhI characterized thus far.

Keywords: Bacillus halodurans ; Characterization; Rhamnose isomerase; Thermostability

Cloning, purification, and characterization of β-galactosidase from Bacillus licheniformis DSM 13 by Onladda Juajun; Thu-Ha Nguyen; Thomas Maischberger; Sanaullah Iqbal; Dietmar Haltrich; Montarop Yamabhai (pp. 645-654).

The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-d-galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K _m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na⁺ and K⁺ in the concentration range of 1–100 mM as well as the divalent metal cations Mg²⁺, Mn²⁺, and Ca²⁺ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.

Keywords: β-Galactosidase; Bacillus licheniformis ; Lactose hydrolysis; Transglycosylation; Recombinant

Nitroreductase activity of ferredoxin reductase BphA4 from Dyella ginsengisoli LA−4 by catalytic and structural properties analysis by Yuanyuan Qu; Hao Zhou; Ang Li; Fang Ma; Jiti Zhou (pp. 655-663).

Ferredoxin reductase BphA4 was well known as a component of biphenyl dioxygenase. However, there was little information about whether it could utilize nonphysiological oxidants as electron acceptors. In the present study, we reported the novel nitroreductase activity of BphA4_LA−4. The homology model of ferredoxin reductase BphA4 from Dyella ginsengisoli LA−4 was constructed. According to the alignment of three-dimensional structures, it was supposed that BphA4_LA−4 could function as nitroreductase. Recombinant His-tagged BphA4_LA−4 was purified with a molecular mass of 49.6 ± 1 kDa. Biochemical characterization of purified BphA4_LA−4 possessed the nitroreductase activity with the optimal temperature 50°C and pH 8.0. The substrate spectrum and kinetics indicated BphA4_LA−4 could reduce several nitroaromatics with different apparent K _m values: m-dinitrobenzene (560 μM), o-dinitrobenzene (1,060 μM), o-nitroaniline (1,570 μM), m-nitrobenzoic acid (1,300 μM) and m-nitrophenol (67 μM). The nitroreductase activity was further explained by docking studies, which was indicated that Arg 288 should play an important role in binding nitroaromatics. Moreover, there existed a good linear correlation between lnK _m and calculated binding energy.

Keywords: Ferredoxin reductase; Nitroreductase; Molecular docking; Homology modeling; Dyella ginsengisoli

Significantly enhanced production of recombinant nitrilase by optimization of culture conditions and glycerol feeding by Jun-Feng Liu; Zhi-Jun Zhang; Ai-Tao Li; Jiang Pan; Jian-He Xu (pp. 665-672).

The production of a recombinant nitrilase expressed in Escherichia coli JM109/pNLE was optimized in the present work. Various culture conditions and process parameters, including medium composition, inducer, induction condition, pH and temperature, were systematically examined. The results showed that nitrilase production in E. coli JM109/pNLE was greatly affected by the pH condition and the temperature in batch culture, and the highest nitrilase production was obtained when the fermentation was carried out at 37°C, initial pH 7.0 without control and E. coli was induced with 0.2 mM isopropyl-β-d-thiogalactoside at 4.0 h. Furthermore, enzyme production could be significantly enhanced by adopting the glycerol feeding strategy with lower flow rate. The enzyme expression was also authenticated by sodium dodecyl phosphate polyacrylamide gel electrophoresis analysis. Finally, under the optimized conditions for fed-batch culture, cell growth, specific activity and nitrilase production of the recombinant E. coli were increased by 9.0-, 5.5-, and 50-fold, respectively.

Keywords: Nitrilase; Optimization; Fermentation; Glycerol feeding; Recombinant Escherichia coli

Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians by Rui Yamaguchi; Hiroko Tokunaga; Matsujiro Ishibashi; Tsutomu Arakawa; Masao Tokunaga (pp. 673-684).

A moderately halophilic bacterium, Kocuria varians, was found to produce active α-amylase (K. varians α-amylase (KVA)). We have observed at least six different forms of α-amylase secreted by this bacterium into the culture medium. Characterization of these KVA forms and cloning of the corresponding gene revealed that KVA comprises pre-pro-precursor form of α-amylase catalytic domain followed by the tandem repeats, which show high similarity to each other and to the starch binding domain (SBD) of other α-amylases. The observed six forms were most likely derived by various processing of the protein product. Recombinant KVA protein was successfully expressed in Escherichia coli as a fusion protein and was purified with affinity chromatography after cleavage from fusion partner. The highly acidic amino acid composition of KVA and the highly negative electrostatic potential surface map of the modeled structure strongly suggested its halophilic nature. Indeed, KVA showed distinct salt- and time-dependent thermal reversibility: when α-amylase was heat denatured at 85°C for 3 min in the presence of 2 M NaCl, the activity was recovered upon incubation on ice (50% recovery after 15 min incubation). Conversely, KVA denatured in 0.1 M NaCl was not refolded at all, even after prolonged incubation. KVA activity was inhibited by proteinaceous α-amylase inhibitor from Streptomyces nitrosporeus, which had been implicated to inhibit only animal α-amylases. KVA with putative SBD regions was found to digest raw starch.

Keywords: Halophilic; Moderate halophile; α-Amylase; Reversibility; α-Amylase inhibitor; Acidic protein

Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii by Elaine O’Connell; Charles Piggott; Maria Tuohy (pp. 685-696).

The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several β-glucan hydrolases including an exo-1,3-β-glucanase. This enzyme was purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different β-glucans, including laminaran, a 1,3-β-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C. At pH 5.0, the enzyme displays strict specificity for laminaran (apparent K _m 1.66 mg mL⁻¹; V _max 7.69 IU mL⁻¹) and laminari-oligosaccharides and did not yield activity against 1,4-β-glucans, 1,3;1,4-β-glucans or 4-nitrophenyl- and methylumbelliferyl-β-d-glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-β-glucanase was inhibited by glucono-δ-lactone (K _i 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as β-glucosidase. ‘De novo’ sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily. The results clearly show that the exo-1,3-β-glucanase is yet another novel enzyme present in the β-glucanolytic enzyme system of T. emersonii.

Keywords: Exo-1,3-β-glucanase; Laminaran; Talaromyces emersonii ; Glucono-δ-lactone; Purification

Identification and characterization of the propanediol utilization protein PduP of Lactobacillus reuteri for 3-hydroxypropionic acid production from glycerol by Lian Hua Luo; Jeong-Woo Seo; Jin-Oh Baek; Baek-Rock Oh; Sun-Yeon Heo; Won-Kyung Hong; Dae-Hyuk Kim; Chul Ho Kim (pp. 697-703).

Although the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol-fermenting microorganisms is still unclear, the propanediol utilization protein (PduP) of Lactobacillus species has been suggested to be a key enzyme in this regard. To verify this hypothesis, a pduP gene from Lactobacillus reuteri was cloned and expressed, and the encoded protein was characterized. Recombinant L. reuteri PduP exhibited broad substrate specificity including 3-hydroxypropionaldehyde and utilized both NAD⁺ and NADP⁺ as a cofactor. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde, at pH 7.8 and 37 °C. The K _m and V _max values for propionaldehyde in the presence of NAD⁺ were 1.18 mM and 0.35 U mg⁻¹, respectively. When L. reuteri pduP was overexpressed in Klebsiella pneumoniae, 3-HP production remarkably increased as compared to the wild-type strain (from 0.18 g L⁻¹ to 0.72 g L⁻¹) under shake-flask culture conditions, and the highest titer (1.38 g L⁻¹ 3-HP) was produced by the recombinant strain under batch fermentation conditions in a bioreactor. This is the first report stating the enzymatic properties of PduP protein and the probable role in biosynthesis of 3-HP in glycerol fermentation.

Keywords: Glycerol; 3-Hydroxypropionic acid; Klebsiella pneumoniae ; Lactobacillus reuteri ; Propanediol utilization protein PduP

Identification of chitinases Is-chiA and Is-chiB from Isoptericola jiangsuensis CLG and their characterization by Ying Wu; Fang Liu; Yi-Chen Liu; Zhen-Hua Zhang; Tian-Tian Zhou; Xin Liu; Qi-Rong Shen; Biao Shen (pp. 705-713).

A 274-bp conserved fragment of chiA (chiA-CF) was amplified from the genomic DNA of Isoptericola jiangsuensis CLG (DSM 21863, CCTCC AB208287) using the specific PCR primers. Based on chiA-CF sequences, a 5233-bp DNA fragment was obtained by self-formed adaptor PCR. DNA sequencing analysis revealed there were two contiguous open reading frames coding for the precursors of Is-chiA [871 amino acids (aa)] and Is-chiB (561 aa) in the 5233-bp DNA fragment. The Is-chiA and Is-chiB exhibited 58% and 62% identity with ArChiA and ArChiB chitinase from Arthrobacter sp. TAD20, respectively. The Is-chiA and Is-chiB genes were cloned into expression vector pET28a (+) and expressed in Escherichia coli BL21 (DE3) with isopropyl-β-d-thiogalactopyranoside induction. Is-chiA and Is-chiB were 92 kDa and 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed chitobiosidase and endochitinase activity, respectively. Is-chiA and Is-chiB were purified by Ni-nitrilotriacetic acid affinity chromatography and the characteristics of both Is-chiA and Is-chiB were studied.

Keywords: Chitinase; Is-chiA and Is-chiB; Cloning and expression; Biological characteristics

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor by Munish Puri; Aneet Kaur; Colin J. Barrow; Ram Sarup Singh (pp. 715-722).

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca⁺² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 g l⁻¹) in the fermentation medium further increased naringinase production (8.9 IU ml⁻¹) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.

Keywords: Citrus peel; Rhamnosidase; Naringin; Bioreactor; Flavonoid

Construction of kanamycin B overproducing strain by genetic engineering of Streptomyces tenebrarius by Xianpu Ni; Dan Li; Lihua Yang; Tingjiao Huang; Hao Li; Huanzhang Xia (pp. 723-731).

Genetic engineering as an important approach to strain optimization has received wide recognition. Recent advances in the studies on the biosynthetic pathways and gene clusters of Streptomyces make stain optimization by genetic alteration possible. Kanamycin B is a key intermediate in the manufacture of the important medicines dibekacin and arbekacin, which belong to a class of antibiotics known as the aminoglycosides. Kanamycin could be prepared by carbamoylkanamycin B hydrolysis. However, carbamoylkanamycin B production in Streptomyces tenebrarius H6 is very low. Therefore, we tried to obtain high kanamycin B-producing strains that produced kanamycin B as a main component. In our work, aprD3 and aprD4 were clarified to be responsible for deoxygenation in apramycin and tobramycin biosynthesis. Based on this information, genes aprD3, aprQ (deduced apramycin biosynthetic gene), and aprD4 were disrupted to optimize the production of carbamoylkanamycin B. Compared with wild-type strain, mutant strain SPU313 (ΔaprD3, ΔaprQ, and ΔaprD4) produced carbamoylkanamycin B as a single antibiotic, whose production increased approximately fivefold. To construct a strain producing kanamycin B instead of carbamoylkanamycin B, the carbamoyl-transfer gene tacA was inactivated in strain SPU313. Mutant strain SPU314 (ΔaprD3, ΔaprQ, ΔaprD4, and ΔtacA) specifically produced kanamycin B, which was proven by LC-MS. This work demonstrated careful genetic engineering could significantly improve production and eliminate undesired products.

Keywords: Strain improvement; Gene disruption; Streptomyces tenebrarius ; Carbamoyl-transfer gene; Deoxygenation; Kanamycin B

The impact of MIG1 and/or MIG2 disruption on aerobic metabolism of succinate dehydrogenase negative Saccharomyces cerevisiae by Hailong Cao; Min Yue; Shuguang Li; Xuefang Bai; Xiaoming Zhao; Yuguang Du (pp. 733-738).

The zinc finger proteins Mig1 and Mig2 play important roles in glucose repression of Saccharomyces cerevisiae. To investigate whether the alleviation of glucose effect would result in an increase in aerobic succinate production, MIG1 and/or MIG2 were disrupted in a succinate dehydrogenase (SDH)-negative S. cerevisiae strain. Moreover, their impacts on physiology of the SDH-negative S. cerevisiae strain were studied under fully aerobic conditions when glucose was the sole carbon source. Our results showed that the succinate production for the SDH-negative S. cerevisiae was very low even under fully aerobic conditions. Furthermore, deletion of MIG1 and/or MIG2 did not result in an increase in succinate production in the SDH-negative S. cerevisiae strain. However, the synthesis of acetate was significantly affected by MIG1 deletion or in combination with MIG2 deletion. The acetate production for the mig1/mig2 double mutant BS2M was reduced by 69.72% compared to the parent strain B2S. In addition, the amount of ethanol produced by BS2M was slightly decreased. With the mig2 mutant BSM2, the concentrations of pyruvate and glycerol were increased by 26.23% and 15.28%, respectively, compared to the parent strain B2S.

Keywords: Saccharomyces cerevisiae ; Succinate dehydrogenase; MIG1 ; MIG2 ; Aerobic metabolism; Glucose repression

Genetic analysis around aminoalcohol dehydrogenase gene of Rhodococcus erythropolis MAK154: a putative GntR transcription factor in transcriptional regulation by Nobuyuki Urano; Michihiko Kataoka; Takeru Ishige; Shinji Kita; Keiji Sakamoto; Sakayu Shimizu (pp. 739-746).

NADP⁺-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 catalyzes the reduction of (S)-1-phenyl-1-keto-2-methylaminopropane ((S)-MAK) to d-pseudoephedrine, which is used as a pharmaceutical. AADH is suggested to participate in aminoalcohol or aminoketone metabolism in this organism because it is induced by the addition of several aminoalcohols, such as 1-amino-2-propanol. Genetic analysis of around the aadh gene showed that some open reading frames (ORFs) are involved in this metabolic pathway. Four of these ORFs might form a carboxysome-like polyhedral organelle, and others are predicted to encode aminotransferase, aldehyde dehydrogenase, phosphotransferase, and regulator protein. OrfE, a homologous ORF of the FadR subfamily of GntR transcriptional regulators, lies downstream from aadh. To investigate whether or not orfE plays a role in the regulation of aadh expression, the gene disruption mutant of R. erythropolis MAK154 was constructed. The ΔorfE strain showed higher AADH activity than wild-type strain. In addition, a transformed strain, which harbored multi-orfE, showed no AADH activity even in the induced condition with 1-amino-2-propanol. These results suggest that OrfE is a negative regulator that represses aadh expression in the absence of 1-amino-2-propanol.

Keywords: Rhodococcus erythropolis ; Aminoalcohol dehydrogenase; Aminoalcohol metabolism; GntR family transcriptional regulator

Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins by Jaewoo Yoon; Jun-ichi Maruyama; Katsuhiko Kitamoto (pp. 747-759).

Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.

Keywords: Aspergillus oryzae ; Decuple protease gene disruption; Heterologous protein production; Bovine chymosin; Human lysozyme; DNA microarray analysis

DNA restriction-modification systems in the ethanologen, Zymomonas mobilis ZM4 by Aidan L. Kerr; Young Jae Jeon; Charles J. Svenson; Peter L. Rogers; Brett A. Neilan (pp. 761-769).

To better understand the DNA restriction-modification (R-M) systems for more amenable strain development of the alternative industrial ethanologen, Zymomonas mobilis, three gene knockout mutants were constructed. The gene knockout mutants were tested for their DNA restriction activities by the determination of transformation efficiency using methylated and unmethylated foreign plasmid DNAs. Inactivation of a putative mrr gene encoded by ZMO0028 (zmrr) resulted in a 60-fold increase in the transformation efficiency when unmethylated plasmid DNA was used. This indicated that the putative mrr gene may serve as a type IV restriction-modification system in Z. mobilis ZM4. To assign the function of a putative type I DNA methyltransferase encoded by ZMO1933 (putative S subunit) and ZMO1934 (putative M subunit), the putative S subunit was inactivated. The gene inactivation of ZMO1933 resulted in a 30-fold increase in the transformation efficiency when methylated plasmid DNA was introduced, indicating that the putative S subunit possibly serves as a part of functional type I R-M system(s). Growth studies performed on the mutant strains indicate inactivation of the type I S subunit resulted in a lower maximum specific glucose consumption rate and biomass yield, while inactivation of the type IV Zmrr had the opposite effect, with an increase in the maximum specific growth rate and biomass yield.

Keywords: Zymomonas mobilis ; Restriction-modification; Homologous recombination; Transformation efficiency; Growth study

Acidithiobacillus thiooxidans secretome containing a newly described lipoprotein Licanantase enhances chalcopyrite bioleaching rate by Roberto A. Bobadilla Fazzini; Gloria Levican; Pilar Parada (pp. 771-780).

The nature of the mineral–bacteria interphase where electron and mass transfer processes occur is a key element of the bioleaching processes of sulfide minerals. This interphase is composed of proteins, metabolites, and other compounds embedded in extracellular polymeric substances mainly consisting of sugars and lipids (Gehrke et al., Appl Environ Microbiol 64(7):2743–2747, 1998). On this respect, despite Acidithiobacilli—a ubiquitous bacterial genera in bioleaching processes (Rawlings, Microb Cell Fact 4(1):13, 2005)—has long been recognized as secreting bacteria (Jones and Starkey, J Bacteriol 82:788–789, 1961; Schaeffer and Umbreit, J Bacteriol 85:492–493, 1963), few studies have been carried out in order to clarify the nature and the role of the secreted protein component: the secretome. This work characterizes for the first time the sulfur (meta)secretome of Acidithiobacillus thiooxidans strain DSM 17318 in pure and mixed cultures with Acidithiobacillus ferrooxidans DSM 16786, identifying the major component of these secreted fractions as a single lipoprotein named here as Licanantase. Bioleaching assays with the addition of Licanantase-enriched concentrated secretome fractions show that this newly found lipoprotein as an active protein additive exerts an increasing effect on chalcopyrite bioleaching rate.

Keywords: Chalcopyrite; Bioleaching; Acidithiobacillus secretome; Metasecretome; Licanantase

Structure-inhibitory activity relationships of pyrrolnitrin analogues on its biosynthesis by Young Soo Keum; Yong-Zhe Zhu; Jeong-Han Kim (pp. 781-789).

Pyrrolnitrin is a bacterial metabolite, served as a natural lead of agricultural fungicides. In a previous study, fenpiclonil was proven to inhibit the oxidative transformation of aminopyrrolnitrin to pyrrolnitrin, catalyzed by aminopyrrolnitrin oxidase (PrnD). This monooxygenase has an interesting catalytic activity of selective oxidation of aromatic amines, rather than aliphatic amines. However, its structural details are not well understood. In this study, various analogues of pyrrolnitrin were prepared to elucidate the structures of active site of PrnD through structure-activity relationships. In vivo pyrrolnitrin biosynthesis inhibition was determined with Burkholderia sp. O33 and Pseudomonas fluorescens Pf-5. Quantitative analysis of pyrrolnitrin and precursors indicates that 2,3-disubstituted phenyl at 3rd carbon and small substituents at 4th carbon of pyrrole are strictly required to give strong inhibitory effects. In addition, dissociable proton of pyrrole is also critical for inhibitory activity. Molecular simulation with homology-based PrnD model suggests a highly restricted conformational space in active site. The results may help more detailed understanding of this unusual enzyme. In addition, the information will be useful for the development of novel fungicide, compatible with pyrrolnitrin-producing bacterium.

Keywords: Pyrrolnitrin; Inhibitor; Structure-activity relationship; Fenpiclonil; Aminopyrrolnitrin oxidase

Non-linear population dynamics in chemostats associated with live–dead cell cycling in Escherichia coli strain K12-MG1655 by Ernest Chi Fru; Irina Dana Ofiţeru; Vasile Lavric; David W. Graham (pp. 791-798).

Bacterial populations conditionally display non-linear dynamic behaviour in bioreactors with steady inputs, which is often attributed to varying habitat conditions or shifting intracellular metabolic activity. However, mathematical modelling has predicted that such dynamics also might simply result from staggered birth, growth, and death events of groups of cells within the population, causing density oscillations and the cycling of live and dead cells within the system. To assess this prediction, laboratory experiments were performed on Escherichia coli strain K12-MG1655 grown in chemostats to first define fine-scale population dynamics over time (minutes) and then determine whether the dynamics correlate with live–dead cell cycles in the system. E. coli populations displayed consistent oscillatory behaviour in all experiments. However, close synchronisation between OD₆₀₀ and live–dead cell oscillations (within ~33–38 min cycles) only became statistically significant (p < 0.01) when pseudo-steady state operations approaching carrying capacity existed in the bioreactor. Specifically, live cells were highest at local OD₆₀₀ maxima and lowest at local OD₆₀₀ minima, showing that oscillations followed live–dead cell cycles as predicted by the model and also consistent with recent observations that death is non-stochastic in such populations. These data show that oscillatory dynamic behaviour is intrinsic in bioreactor populations, which has implications to process operations in biotechnology.

Keywords: Bacteria; Chemostats; Population dynamics; Cell death; Carrying capacity

Interaction of SCO2127 with BldKB and its possible connection to carbon catabolite regulation of morphological differentiation in Streptomyces coelicolor by Adán Chávez; Angela Forero; Mauricio Sánchez; Romina Rodríguez-Sanoja; Guillermo Mendoza-Hernández; Luis Servín-Gonzalez; Brenda Sánchez; Yolanda García-Huante; Diana Rocha; Elizabeth Langley; Beatriz Ruiz; Sergio Sánchez (pp. 799-806).

In Streptomyces coelicolor, the sco2127 gene is located upstream of the gene encoding for glucose kinase. This region restores sensitivity to carbon catabolite repression (CCR) of Streptomyces peucetius var. caesius mutants, resistant to 2-deoxyglucose (Dog^R). In order to search for the possible mechanisms behind this effect, sco2127 was overexpressed and purified for protein–protein interaction studies. SCO2127 was detected during the late growth phase of S. coelicolor grown in a complex media supplemented with 100 mM glucose. Pull-down assays using crude extracts from S. coelicolor grown in the same media, followed by far-western blotting, allowed detection of two proteins bound to SCO2127. The proteins were identified by MALDI-TOF mass spectrometry as SCO5113 and SCO2582. SCO5113 (BldKB) is a lipoprotein ABC-type permease (∼66 kDa) involved in mycelium differentiation by allowing the transport of the morphogenic oligopeptide Bld261. SCO2582, is a putative membrane metalloendopeptidase (∼44 kDa) of unknown function. In agreement with the possible role of SCO2127 in mycelium differentiation, delayed aerial mycelium septation and sporulation was observed when S. coelicolor A3(2) was grown in the presence of elevated glucose concentrations (100 mM), an effect not seen in a Δ-sco2127 mutant derived from it. We speculate that SCO2127 might represent a key factor in CCR of mycelium differentiation by interacting with BldKB.

Keywords: Streptomyces coelicolor ; SCO2127; Carbon catabolite repression; BldKB; SCO2582; Protein–protein interactions

Evaluation of different sources of DNA for use in genome wide studies and forensic application by Habiba S. Al Safar; Fatima H. Abidi; Kamal A. Khazanehdari; Ian R. Dadour; Guan K. Tay (pp. 807-815).

In the field of epidemiology, Genome-Wide Association Studies (GWAS) are commonly used to identify genetic predispositions of many human diseases. Large repositories housing biological specimens for clinical and genetic investigations have been established to store material and data for these studies. The logistics of specimen collection and sample storage can be onerous, and new strategies have to be explored. This study examines three different DNA sources (namely, degraded genomic DNA, amplified degraded genomic DNA and amplified extracted DNA from FTA card) for GWAS using the Illumina platform. No significant difference in call rate was detected between amplified degraded genomic DNA extracted from whole blood and amplified DNA retrieved from FTA™ cards. However, using unamplified–degraded genomic DNA reduced the call rate to a mean of 42.6% compared to amplified DNA extracted from FTA card (mean of 96.6%). This study establishes the utility of FTA™ cards as a viable storage matrix for cells from which DNA can be extracted to perform GWAS analysis.

Keywords: FTA; GWAS; DNA quality

A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry by Yu-Cai He; Cui-Luan Ma; Jian-He Xu; Li Zhou (pp. 817-823).

Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.

Keywords: Nitrilase; Carboxylic acids; High-throughput screening; Ferric hydroxamate spectrophotometry; Evaluation

Increased bioclogging and corrosion risk by sulfate addition during iodine recovery at a natural gas production plant by Choon-Ping Lim; Dan Zhao; Yuta Takase; Kazuhiko Miyanaga; Tomoko Watanabe; Yasuyoshi Tomoe; Yasunori Tanji (pp. 825-834).

Iodine recovery at a natural gas production plant in Japan involved the addition of sulfuric acid for pH adjustment, resulting in an additional about 200 mg/L of sulfate in the waste brine after iodine recovery. Bioclogging occurred at the waste brine injection well, causing a decrease in well injectivity. To examine the factors that contribute to bioclogging, an on-site experiment was conducted by amending 10 L of brine with different conditions and then incubating the brine for 5 months under open air. The control case was exposed to open air but did not receive additional chemicals. When sulfate addition was coupled with low iodine, there was a drastic increase in the total amount of accumulated biomass (and subsequently the risk of bioclogging) that was nearly six times higher than the control. The bioclogging-associated corrosion rate of carbon steel was 84.5 μm/year, which is four times higher than that observed under other conditions. Analysis of the microbial communities by denaturing gradient gel electrophoresis revealed that the additional sulfate established a sulfur cycle and induced the growth of phototrophic bacteria, including cyanobacteria and purple bacteria. In the presence of sulfate and low iodine levels, cyanobacteria and purple bacteria bloomed, and the accumulation of abundant biomass may have created a more conducive environment for anaerobic sulfate-reducing bacteria. It is believed that the higher corrosion rate was caused by a differential aeration cell that was established by the heterogeneous distribution of the biomass that covered the surface of the test coupons.

Keywords: Bioclogging; Microbiologically influenced corrosion; Sulfate-reducing bacteria; Purple bacteria; Sulfur cycle; Natural gas field

Bacterial community analysis of swine manure treated with autothermal thermophilic aerobic digestion by Il Han; Shankar Congeevaram; Dong-Won Ki; Byoung-Taek Oh; Joonhong Park (pp. 835-842).

Due to the enviornmental problems associated with disposal of livestock sludge, many stabilization studies emphasizing on the sludge volume reduction were performed. However, little is known about the microbial risk present in sludge and its stabilized products. This study microbiologically explored the effects of anaerobic lagoon fermentation (ALF) and autothermal thermophilic aerobic digestion (ATAD) on pathogen-related risk of raw swine manure by using culture-independent 16S rDNA cloning and sequencing methods. In raw swine manure, clones closely related to pathogens such as Dialister pneumosintes, Erysipelothrix rhusiopathiae, Succinivibrioan dextrinosolvens, and Schineria sp. were detected. Meanwhile, in the mesophilic ALF-treated swine manure, bacterial community clones closely related to pathogens such as Schineria sp. and Succinivibrio dextrinosolvens were still detected. Interestingly, the ATAD treatment resulted in no detection of clones closely related to pathogens in the stabilized thermophilic bacterial community, with the predominance of novel Clostridia class populations. These findings support the superiority of ATAD in selectively reducing potential human and animal pathogens compared to ALF, which is a typical manure stabilization method used in livestock farms.

Keywords: Livestock sludge; Human and animal pathogens; Livestock sludge stabilization; Autothermal thermophilic aerobic digestion; Microbial community analysis

Change in ammonia-oxidizing microorganisms in enriched nitrifying activated sludge by Puntipar Sonthiphand; Tawan Limpiyakorn (pp. 843-853).

In this study, sludge was taken from a municipal wastewater treatment plant that contained a nearly equal number of archaeal amoA genes (5.70 × 10⁶ ± 3.30 × 10⁵ copies mg sludge⁻¹) to bacterial amoA genes (8.60 × 10⁶ ± 7.64 × 10⁵ copies mg sludge⁻¹) and enriched in three continuous-flow reactors receiving an inorganic medium containing different ammonium concentrations: 2, 10, and 30 mM NH ₄ ⁺ –N (28, 140, and 420 mg N l⁻¹). The abundance and communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in enriched nitrifying activated sludge (NAS) were monitored at days 60 and 360 of the operation. Early on, between day 0 and day 60 of reactor operation, comparative abundance of AOA amoA genes to AOB amoA genes varied among the reactors depending on the ammonium levels found in the reactors. As compared to the seed sludge, the number of AOA amoA genes was unchanged in the reactor with lower ammonium level (0.06 ± 0.04 mgN l⁻¹), while in the reactors with higher ammonium levels (0.51 ± 0.33 and 0.25 ± 0.10 mgN l⁻¹), the numbers of AOA amoA genes were deteriorated. By day 360, AOA disappeared from the ammonia-oxidizing consortiums in all reactors. The majority of the AOA sequences from all NASs at each sampling period fell into a single AOA cluster, however, suggesting that the ammonium did not affect the AOA communities under this operational condition. This result is contradictory to the case of AOB, where the communities varied significantly among the NASs. AOB with a high affinity for ammonia were present in the reactors with lower ammonium levels, whereas AOB with a low affinity to ammonia existed in the reactors with higher ammonium levels.

Keywords: Ammonia-oxidizing archaea; Ammonia-oxidizing bacteria; amoA gene; Nitrifying activated sludge; Wastewater

Upgrading of straw hydrolysate for production of hydrogen and phenols in a microbial electrolysis cell (MEC) by Anders Thygesen; Massimo Marzorati; Nico Boon; Anne Belinda Thomsen; Willy Verstraete (pp. 855-865).

In a microbial electrolysis cell (MEC), hydrolysate produced by hydrothermal treatment of wheat straw was used for hydrogen production during selective recovery of phenols. The average H₂ production rate was 0.61 m³ H₂/m³ MEC·day and equivalent to a rate of 0.40 kg COD/m³ MEC·day. The microbial community in the anode biofilm was adapted by establishment of xylose-degrading bacteria of the Bacteriodetes phylum (16%) and Geobacter sulfurreducens (49%). During the process, 61% of the chemical oxygen demand was removed as hydrogen at 64% yield. The total energy production yield was 78% considering the energy content in the consumed compounds and the cell voltage of 0.7 V. The highest hydrogen production was equivalent to 0.8 kg COD/m³ MEC·day and was obtained at pH 7–8 and 25°C. Accumulation of 53% w/v phenolic compounds in the liquor was obtained by stepwise addition of the hydrolysate during simultaneous production of hydrogen from consumption of 95% for the hemicellulose and 100% of the fatty acids. Final calculations showed that hydrolysate produced from 1 kg wheat straw was upgraded by means of the MEC to 22 g hydrogen (266 L), 8 g xylan, and 9 g polyphenolics for potential utilization in biobased materials.

Keywords: Wheat straw; Hydrothermal treatment; Hemicellulose; Phenolic compounds; Hydrogen

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Applied Microbiology and Biotechnology (v.89, #3)

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Applied Microbiology and Biotechnology (v.89, #3)