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Applied Microbiology and Biotechnology (v.89, #4)

Genetically engineered microbial biosensors for in situ monitoring of environmental pollution by Hae Ja Shin (pp. 867-877).

Microbial biosensors are compact, portable, cost effective, and simple to use, making them seem eminently suitable for the in situ monitoring of environmental pollution. One promising approach for such applications is the fusion of reporter genes with regulatory genes that are dose-dependently responsive to the target chemicals or physiological signals. Their biosensor capabilities, such as target range and sensitivity, could be improved by modification of regulatory genes. Recent uses of such genetically engineered microbial biosensors include the development of portable biosensor kits and high-throughput cell arrays on chips, optic fibers, or other platforms for on-site and on-line monitoring of environmental pollution. This mini-review discusses recent advances in microbial biosensors and their future prospects, with a focus on the development and application of genetically modified microbial biosensors for in situ environmental monitoring.

Keywords: Microbial biosensor; Genetically engineered; In situ environmental monitoring

Biotechnological production of mannitol and its applications by Badal C. Saha; F. Michael Racine (pp. 879-891).

Mannitol, a naturally occurring polyol (sugar alcohol), is widely used in the food, pharmaceutical, medical, and chemical industries. The production of mannitol by fermentation has become attractive because of the problems associated with its production chemically. A number of homo- and heterofermentative lactic acid bacteria (LAB), yeasts, and filamentous fungi are known to produce mannitol. In particular, several heterofermentative LAB are excellent producers of mannitol from fructose. These bacteria convert fructose to mannitol with 100% yields from a mixture of glucose and fructose (1:2). Glucose is converted to lactic acid and acetic acid, and fructose is converted to mannitol. The enzyme responsible for conversion of fructose to mannitol is NADPH- or NADH-dependent mannitol dehydrogenase (MDH). Fructose can also be converted to mannitol by using MDH in the presence of the cofactor NADPH or NADH. A two enzyme system can be used for cofactor regeneration with simultaneous conversion of two substrates into two products. Mannitol at 180 g l⁻¹ can be crystallized out from the fermentation broth by cooling crystallization. This paper reviews progress to date in the production of mannitol by fermentation and using enzyme technology, downstream processing, and applications of mannitol.

Keywords: Mannitol production; Homofermentative lactic acid bacterium; Heterofermentative lactic acid bacterium; Fermentation; Mannitol dehydrogenase

Development and strategies of cell-culture technology for influenza vaccine by Shao-Zhen Feng; Pei-Rong Jiao; Wen-Bao Qi; Hui-Ying Fan; Ming Liao (pp. 893-902).

Influenza is a pandemic contagious disease and causes human deaths and huge economic destruction of poultry in the world. In order to control and prevent influenza, mainly type A, influenza vaccine for human and poultry were available since the 1940s and 1920s, respectively. In the development of vaccine production, influenza viruses were cultured originally from chicken embryos to anchorage-dependent cell lines, such as MDCK and Vero. The anchorage-independent lines have also been used to produce influenza virus, such as PER.C6 and engineering modified MDCK and Vero. During the process of influenza vaccine production, the common problem faced by all producers is how to improve the titer of influenza virus. This paper focuses on the developments of cell culture for influenza virus vaccine production, limitations of cell culture, and relative strategies for improvement virus yields in cell-culture systems.

Keywords: Cell culture; Development; Influenza virus; Strategies; Vaccine

Bacterial degradation of bile salts by Bodo Philipp (pp. 903-915).

Bile salts are surface-active steroid compounds. Their main physiological function is aiding the digestion of lipophilic nutrients in intestinal tracts of vertebrates. Many bacteria are capable of transforming and degrading bile salts in the digestive tract and in the environment. Bacterial bile salt transformation and degradation is of high ecological relevance and also essential for the biotechnological production of steroid drugs. While biotechnological aspects have been reviewed many times, the physiological, biochemical and genetic aspects of bacterial bile salt transformation have been neglected. This review provides an overview of the reaction sequence of bile salt degradation and on the respective enzymes and genes exemplified with the degradation pathway of the bile salt cholate. The physiological adaptations for coping with the toxic effects of bile salts, recent biotechnological applications and ecological aspects of bacterial bile salt metabolism are also addressed. As the pathway for bile salt degradation merges with metabolic pathways for bacterial transformation of other steroids, such as testosterone and cholesterol, this review provides helpful background information for metabolic engineering of steroid-transforming bacteria in general.

Keywords: Biodegradation; Steroids; Bile salts

Interactions between arbuscular mycorrhizal fungi and soil bacteria by Mohammad Miransari (pp. 917-930).

The soil environment is interesting and complicated. There are so many interactions taking place in the soil, which determine the properties of soil as a medium for the growth and activities of plants and soil microorganisms. The soil fungi, arbuscular mycorrhiza (AM), are in mutual and beneficial symbiosis with most of the terrestrial plants. AM fungi are continuously interactive with a wide range of soil microorganisms including nonbacterial soil microorganisms, plant growth promoting rhizobacteria, mycorrhiza helper bacteria and deleterious bacteria. Their interactions can have important implications in agriculture. There are some interesting interactions between the AM fungi and soil bacteria including the binding of soil bacteria to the fungal spore, the injection of molecules by bacteria into the fungal spore, the production of volatiles by bacteria and the degradation of fungal cellular wall. Such mechanisms can affect the expression of genes in AM fungi and hence their performance and ecosystem productivity. Hence, consideration of such interactive behavior is of significance. In this review, some of the most important findings regarding the interactions between AM fungi and soil bacteria with some new insights for future research are presented.

Keywords: Arbuscular mycorrhizal fungi; Beneficial and deleterious effects; Soil bacteria; Agricultural implications

Exploitation of phage battery in the search for bioactive actinomycetes by D. İpek Kurtböke (pp. 931-937).

Screening of microbial natural products continues to represent an important route to the discovery of novel bioactive compounds for the development of new therapeutic agents, and actinomycetes are still the major producers of biopharmaceuticals. Selective isolation of bioactive actinomycete species, in particular the rare ones, has thus become a target for industrial microbiologists. In this context, bacteriophages have proven to be useful tools as (1) naturally present indicators of under-represented or rare actinomycete taxa in environmental samples, (2) indicators of the relatedness of bioactive taxa in target-directed search and discovery, (3) de-selection agents of unwanted taxa on isolation plates in target-specific search for rare actinomycete taxa, (4) tools in screening assays for specific targets. Against this background, a number of case studies are presented to illustrate the use of bacteriophages as tools in actinomycete-origin bioactive compound search and discovery programs.

Keywords: Actinomycetes; Bioactive compounds; Bacteriophages; Selective isolation of rare actinomycetes

Production of recombinant proteins and metabolites in yeasts by Danilo Porro; Brigitte Gasser; Tiziana Fossati; Michael Maurer; Paola Branduardi; Michael Sauer; Diethard Mattanovich (pp. 939-948).

Recombinant DNA (rDNA) technologies allow the production of a wide range of peptides, proteins and metabolites from naturally non-producing cells. Since human insulin was the first heterologous compound produced in a laboratory in 1977, rDNA technology has become one of the most important technologies developed in the 20th century. Recombinant protein and metabolites production is a multi-billion dollar market. The development of a new product begins with the choice of the cell factory. The final application of the compound dictates the main criteria that should be taken into consideration: (1) quality, (2) quantity, (3) yield and (4) space time yield of the desired product. Quantity and quality are the most predominant requirements that must be considered for the commercial production of a protein. Quantity and yield are the requirements for the production of a metabolite. Finally, space time yield is crucial for any production process. It therefore becomes clear why the perfect host does not exist yet, and why—despite important advances in rDNA applications in higher eukaryotic cells—microbial biodiversity continues to represent a potential source of attractive cell factories. In this review, we compare the advantages and limitations of the principal yeast and bacterial workhorse systems.

Keywords: Recombinant proteins; Recombinant metabolites; Bacterial host systems; Yeast host systems; Industrial biotechnology

Aspartic proteinases from Mucor spp. in cheese manufacturing by Sirma Yegin; Marcelo Fernandez-Lahore; Antonio Jose Gama Salgado; Ulgar Guvenc; Yekta Goksungur; Canan Tari (pp. 949-960).

Filamentous fungi belonging to the order of Mucorales are well known as producers of aspartic proteinases depicting milk-clotting activity. The biosynthesis level, the biochemical characteristics, and the technological properties of the resulting proteinases are affected by the producer strain and the mode of cultivation. While the milk-clotting enzymes produced by the Rhizomucor spp. have been extensively studied in the past, much less is known on the properties and potential applications of the aspartic proteinases obtained for Mucor spp. Indeed, several Mucor spp. strains have been reported as a potential source of milk-clotting enzymes having unique technological properties. Both submerged fermentation and solid substrate cultivation are proven alternatives for the production of Mucor spp. aspartic proteinases. This review provides an overview on the bioprocessing routes to obtain large amounts of these enzymes, on their structural characteristics as related to their functional properties, and on their industrial applications with focus on cheese manufacturing.

Keywords: Mucor spp. proteinase; Aspartic proteinase; Acid protease; Milk-clotting enzymes

Current knowledge of microbial community structures in landfills and its cover soils by Jeremy D. Semrau (pp. 961-969).

Landfills are a vital component of our waste handling processes. Our lack of knowledge on the microbial processes in these systems, however, hampers our ability to design the next generation of landfills that: (1) enhance the rate and extent of waste decomposition, (2) produce byproducts of some value (e.g., methane that can be used for energy generation), and, (3) minimize their overall impact on driving climate change through the emission of greenhouse gases. In this review, the current state of knowledge the microbial community structure and activity in both the refuse and overlying cover soils is discussed, and suggestions provided for future research in this critical aspect of our infrastructure.

Keywords: Landfill; Methanogenesis; Methanotroph; Ammonia-oxidizing microorganisms; Celluloytic microorganisms; Acidogens

Production of edible mushrooms in forests: trends in development of a mycosilviculture by Jean-Michel Savoie; Michèle L. Largeteau (pp. 971-979).

Developing the production of ectomycorrhizal (ECM) mushrooms in forest has become a challenge. Only a few ECM species are currently cultivable. Controlled mycorrhization practices offer promising advance to produce currently uncultivable ECM mushrooms. The persistence of the production of edible species, either cultivated or wild, depends on both the tree and the ecological environment (fungal communities, climate, soil, tree development). Developing adapted forest management practices appears to be means to improve production of edible ECM mushrooms. This review summarises current knowledge on the development of a science-based mycosilviculture for the production of edible ECM mushrooms.

Keywords: Controlled mycorrhization; Ectomycorrhizal mushrooms; Forest management; Fungal succession

Polyoxometalate/laccase-mediated oxidative polymerization of catechol for textile dyeing by Suyeon Kim; Carla Silva; Dmitry V. Evtuguin; José A. F. Gamelas; Artur Cavaco-Paulo (pp. 981-987).

The synergistic effect between polyoxometalates (POMs), namely K₅[SiW₁₁V^VO₄₀]·11H₂O and H₅[PMo₁₀V^V ₂O₄₀]·13H₂O and laccase from ascomycete Myceliophthora thermophila has been employed for the first time in oxidative polymerization of catechol. Such a laccase-mediator system allowed the formation of a relatively high molecular weight polycatechol as confirmed by size exclusion chromatography and electrospray ionization mass spectrometry (ESI-MS) (3990 Da when using K₅[SiW₁₁V^VO₄₀]·11H₂O and 3600 Da with H₅[PMo₁₀V^V ₂O₄₀]·13H₂O). The synthesized polymers were applied as dyes for the dyeing of flax fabrics. The color intensity of flax fabrics colored with polymer solutions was evaluated by diffuse reflectance spectrophotometry via k/s measurements (+10% of fixation ratio). A new synthetic process allowed a dyeing polymer, provided upon flax coloration, better color fixation and color resistance when compared to that obtained by conventional synthesis with laccase solely or with addition of organic mediator (1-hydroxybenzotriazole).

Keywords: Polyoxometalate; Laccase; Catechol; Mediator; Polymerization

Production of tranilast [N-(3′,4′-dimethoxycinnamoyl)-anthranilic acid] and its analogs in yeast Saccharomyces cerevisiae by Aymerick Eudes; Edward E. K. Baidoo; Fan Yang; Helcio Burd; Masood Z. Hadi; F. William Collins; Jay D. Keasling; Dominique Loqué (pp. 989-1000).

Biological synthesis of therapeutic drugs beneficial for human health using microbes offers an alternative production strategy to the methods that are commonly employed such as direct extraction from source organisms or chemical synthesis. In this study, we evaluated the potential for yeast (Saccharomyces cerevisiae) to be used as a catalyst for the synthesis of tranilast and various tranilast analogs (cinnamoyl anthranilates). Several studies have demonstrated that these phenolic amides have antioxidant properties and potential therapeutic benefits including antiinflammatory, antiproliferative, and antigenotoxic effects. The few cinnamoyl anthranilates naturally produced in plants such as oats and carnations result from the coupling of various hydroxycinnamoyl-CoAs to anthranilic acid. In order to achieve the microbial production of tranilast and several of its analogs, we engineered a yeast strain to co-express a 4-coumarate/CoA ligase (4CL, EC 6.2.1.12) from Arabidopsis thaliana and a hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT, EC 2.3.1.144) from Dianthus caryophyllus. This modified yeast strain allowed us to produce tranilast and 26 different cinnamoyl anthranilate molecules within a few hours after exogenous supply of various combinations of cinnamic acids and anthranilate derivatives. Our data demonstrate the feasibility of rapidly producing a wide range of defined cinnamoyl anthranilates in yeast and underline a potential for the biological designed synthesis of naturally and non-naturally occurring molecules.

Keywords: Cinnamoyl anthranilate; Tranilast; Avenanthramides; Recombinant yeast; HCBT

Production of indole-3-acetic acid and related indole derivatives from L-tryptophan by Rubrivivax benzoatilyticus JA2 by Md. Mujahid; Ch. Sasikala; Ch. V. Ramana (pp. 1001-1008).

Rubrivivax benzoatilyticus JA2 produces indoles with simultaneous utilization of L-tryptophan. Fifteen chromatographically distinct indole derivatives were detected from the L-tryptophan-supplemented cultures of R. benzoatilyticus JA2. Nine of these were identified as, indole 3-acetamide, Methoxyindole-3-aldehyde, indole 3-aldehyde, methoxyindole-3-acetic acid, indole 3-acetic acid, indole-3-carboxylic acid, indole-3-acetonitrile, indole, and trisindoline. Tryptophan stable isotope feeding confirmed the indoles produced are from the supplemented L-tryptophan. Indole 3-acetic acid is one of the major products of L-tryptophan catabolism by R. benzoatilyticus JA2 and its production was influenced by growth conditions. Identification of indole 3-acetamide and tryptophan monooxygenase activity suggests indole 3-acetamide routed IAA biosynthesis in R. benzoatilyticus JA2. The study also indicated the possible multiple pathways of IAA biosynthesis in R. benzoatilyticus JA2.

Keywords: Rubrivivax benzoatilyticus ; Indole 3-acetic acid; Indole; L-Tryptophan; Catabolism; Metabolite probe

Highly efficient production of d-lactate by Sporolactobacillus sp. CASD with simultaneous enzymatic hydrolysis of peanut meal by Limin Wang; Bo Zhao; Fengsong Li; Ke Xu; Cuiqing Ma; Fei Tao; Qinggang Li; Ping Xu (pp. 1009-1017).

Highly efficient d-lactate production by Sporolactobacillus sp. strain CASD was demonstrated in this study. Peanut meal was found to be a better nutrient than yeast extract, soybean meal, soybean peptone, corn steep, liquor beef extract, and ammonium sulfate in the production of d-lactate. To improve the utilization of peanut meal, the material was enzymatically hydrolyzed and simultaneously utilized as the nitrogen source in d-lactate fermentation. Very high d-lactate production (207 g/L) was obtained using 40 g/L of peanut meal in 30-L fed-batch fermentation, with the average productivity of 3.8 g/(L·h) and optical purity of 99.3%. The production of such a high concentration of optically pure d-lactate by strain CASD, with the simultaneous enzymatic hydrolysis of peanut meal and fermentation, represents a new cost-efficient and integrated method for d-lactate production using agricultural by-products.

Keywords: d-Lactate; Sporolactobacillus sp.; Peanut meal; Simultaneous enzymatic hydrolysis

Production of aglycon protopanaxadiol via compound K by a thermostable β-glycosidase from Pyrococcus furiosus by Mi-Hyun Yoo; Soo-Jin Yeom; Chang-Su Park; Ki-Won Lee; Deok-Kun Oh (pp. 1019-1028).

The production of compound K and aglycon protopanaxadiol (APPD) from ginsenoside Rd and ginseng root extract was performed using a recombinant β-glycosidase from Pyrococcus furiosus. The activity for Rd was optimal at pH 5.5 and 95°C with a half-life of 68 h at 95°C. β-Glycosidase converted Rb₁, Rb₂, Rc, and Rd to APPD via compound K. With increases in the enzyme activity, the productivities of compound K and APPD increased. The substrate concentration was optimal at 4.0 mM Rd or 10% (w/v) ginseng root extract; 4 mM of Rd was converted to 3.3 mM compound K with a yield of 82.5% (mol/mol) and a productivity of 2,010 mg l⁻¹ h⁻¹ at 1 h and was hydrolyzed completely to APPD with 364 mg l⁻¹ h⁻¹ after 5 h. Rb₁, Rb₂, Rc, and Rd at 3.9 mM in 10% ginseng root extract were converted to 3.1 mM compound K with 79.5% and 1,610 mg l⁻¹ h⁻¹ at 1.2 h and were hydrolyzed completely to APPD with 300 mg l⁻¹ h⁻¹ after 6 h. The concentrations and productivities of compound K and APPD in the present study are the highest ever reported.

Keywords: Aglycon protopanaxadiol; Compound K; β-glycosidase; Pyrococcus furiosus

Metabolic changes underlying the higher accumulation of glutathione in Saccharomyces cerevisiae mutants by Ildar Nisamedtinov; Kaspar Kevvai; Kerti Orumets; Liisa Arike; Inga Sarand; Matti Korhola; Toomas Paalme (pp. 1029-1037).

Molecular mechanisms leading to glutathione (GSH) over-accumulation in a Saccharomyces cerevisiae strain produced by UV irradiation-induced random mutagenesis were studied. The mutant accumulated GSH but also cysteine and γ-glutamylcysteine in concentrations that were several fold higher than in its wild-type parent strain under all studied cultivation conditions (chemostat, fed-batch, and turbidostat). Transcript analyses along with shotgun proteome quantification indicated a difference in the expression of a number of genes and proteins, the most pronounced of which were several fold higher expression of CYS3, but also that of GSH1 and its transcriptional activator YAP1. This together with the higher intracellular cysteine concentration is most likely the primary factor underlying GSH over-accumulation in the mutant. Comparative sequencing of GSH1 and the fed-batch experiments with continuous cysteine addition demonstrated that the feedback inhibition of Gsh1p by GSH was still operational in the mutant.

Keywords: Saccharomyces cerevisiae ; Glutathione; Cysteine; GSH1 ; CYS3 ; YAP1

Isolation and characterisation of lactic acid bacterium for effective fermentation of cellobiose into optically pure homo l-(+)-lactic acid by Mohamed Ali Abdel-Rahman; Yukihiro Tashiro; Takeshi Zendo; Keisuke Shibata; Kenji Sonomoto (pp. 1039-1049).

Effective utilisation of cellulosic biomasses for economical lactic acid production requires a microorganism with potential ability to utilise efficiently its major components, glucose and cellobiose. Amongst 631 strains isolated from different environmental samples, strain QU 25 produced high yields of l-(+)-lactic acid of high optical purity from cellobiose. The QU 25 strain was identified as Enterococcus mundtii based on its sugar fermentation pattern and 16S rDNA sequence. The production of lactate by fermentation was optimised for the E. mundtii QU25 strain. The optimal pH and temperature for batch culturing were found to be 7.0°C and 43°C, respectively. E. mundtii QU 25 was able to metabolise a mixture of glucose and cellobiose simultaneously without apparent carbon catabolite repression. Moreover, under the optimised culture conditions, production of optically pure l-lactic acid (99.9%) increased with increasing cellobiose concentrations. This indicates that E. mundtii QU 25 is a potential candidate for effective lactic acid production from cellulosic hydrolysate materials.

Keywords: l-Lactic acid production; Glucose; Cellobiose; Mixed sugars; Enterococcus mundtii

Cloning and characterisation of a cystathionine β/γ-lyase from two Oenococcus oeni oenological strains by Caroline Knoll; Maret du Toit; Sylvia Schnell; Doris Rauhut; Stefan Irmler (pp. 1051-1060).

Sulphur-containing compounds in wine have been extensively studied because of their effect on wine flavour and quality. In this study, an enzyme that degrades sulphur-containing amino acids was cloned and characterised from two Oenococcus oeni strains of oenological origins. The enzyme has features of a cystathionine-γ-lyase (EC 4.4.1.1), a pyridoxal-5-phosphate-dependent enzyme catalysing an α,γ-elimination reaction of l-cystathionine to produce l-cysteine, α-ketobutyrate and ammonia. Moreover, it was able to catalyse an α,β-elimination reaction producing homocysteine, pyruvate and ammonia from l-cystathionine. An elimination reaction of l-cysteine and dl-homocysteine was also efficiently catalysed by the enzyme, resulting in the formation of hydrogen sulphide. Furthermore, the ability to demethiolate methionine into methanethiol, an unfavourable volatile sulphur compound in terms of wine aroma, was observed. The findings of this work suggest that O. oeni seems to play a minor role in the production of volatile sulphur compounds during the vinification process as the optimal conditions were far from the harsh wine environment.

Keywords: Oenococcus oeni ; Cystathionine lyase; Volatile sulphur compounds; Enzyme activity

Functional characterization of a new holin-like antibacterial protein coding gene tmp1 from goat skin surface metagenome by Thangamani Rajesh; Thangamani Anthony; Subramani Saranya; Paul Lavanya Pushpam; Paramasamy Gunasekaran (pp. 1061-1073).

We have identified a holin-like gene from a goat skin surface metagenome. The ORF designated tmp1 coding for 34 amino acids shared sequence similarity with putative holin-like toxin genes. To analyze the antibacterial activity of tmp1 encoded protein, this ORF was cloned and expressed in Escherichia coli BL21(DE3). The expressed gene product Tmp1 exhibited antibacterial activity against Gram-positive bacteria but not to Gram-negative bacteria. A single transmembrane domain (TMD) was identified within Tmp1 and deletion analysis of the N-terminal region and TMD indicated TMD to be responsible for antibacterial activity. The TMD-dependent antibacterial activity was validated using a synthetic peptide with the amino acid sequence of TMD. Besides antibacterial activity, Tmp1 also complemented the function of holin in a lysis-defective bacteriophage lambda. To broaden the spectrum of antibacterial activity, a mutant library of tmp1 was generated by random mutagenesis. Four mutants with amino acid substitutions at the N-terminus of Tmp1 exhibited increased antibacterial activity against Gram-positive and Gram-negative bacteria and were not hemolytic. An improved activity of these mutant proteins is attributed to their increased hydrophobicity.

Keywords: Metagenome; Holin-like protein; Antibacterial activity; Transmembrane domain; Site-directed mutagenesis; Hemolytic activity

Characterization of a novel dye-linked l-proline dehydrogenase from an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis by Takenori Satomura; Xiao-Dong Zhang; Yusuke Hara; Katsumi Doi; Haruhiko Sakuraba; Toshihisa Ohshima (pp. 1075-1082).

The activity of a dye-linked l-proline dehydrogenase (dye-l-proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about 108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5–9.0 for 30 min at 50 °C. The enzyme catalyzed l-proline dehydrogenation to Δ¹-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for l-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-l-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far.

Keywords: l-Proline dehydrogenase; FAD containing amino acid dehydrogenase; Hyperthermophilic archaeon; Pyrobaculum calidifontis

Cloning and functional characterization of a novel endo-β-1,4-glucanase gene from a soil-derived metagenomic library by Juan Liu; Wei-dong Liu; Xiao-li Zhao; Wen-jing Shen; Hui Cao; Zhong-li Cui (pp. 1083-1092).

A metagenomic library consisting of 3,024 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B with high-molecular-weight DNA extracted from red soil in South China. A novel cellulase gene with an open reading frame of 1,332 bp, cel5G, encoding an endo-β-1,4-glucanase was cloned using an activity-based screen. The deduced enzyme, Cel5G, belongs to the glycosyl hydrolase family 5 and shares <39% identity with endoglucanases in the GenBank database. cel5G was expressed in E. coli BL21, and the recombinant enzyme Cel5G was purified to homogeneity for enzymatic analysis. Cel5G hydrolyzed a wide range of β-1,4-, β-1,3/β-1,4-, or β-1,3/β-1,6-linked polysaccharides, amorphous cellulose, filter paper, and microcrystalline cellulose. Its highest activity was in 50 mM citrate buffer, pH 4.8, at 50°C. Cel5G is stable over a wide range of pH values (from 2.0 to 10.6) and is thermally stable under 60°C. It is highly tolerant and active in high salt concentrations and is stable in the presence of pepsin and pancreatin. The K _m and V _max values of Cel5G for carboxymethyl cellulose are 19.92 mg/ml and 1,941 μmol min⁻¹ mg⁻¹, respectively. These characteristics indicate that Cel5G has potential for industrial use.

Keywords: Metagenomic BAC library; Cellulase; Endo-β-1,4-glucanase; Gene cloning; Functional characterization

Recombinant expression, affinity purification and functional characterization of Scots pine defensin 1 by Valentina Kovaleva; Hryhoriy Krynytskyy; Ivan Gout; Roman Gout (pp. 1093-1101).

Plants produce a variety of molecules to defend themselves from fungal pathogens. Defensins belong to the family of antimicrobial peptides that play a central role in innate immunity in all species of plants. We have previously reported the purification of antimicrobial peptides from Scots pine seedlings and the identification of some of them, including defensin, by mass spectrometry. In this study, we extend our original study on molecular cloning of Pinus sylvestris defensin 1 (PsDef1) by presenting the expression and affinity purification of recombinant defensin 1 (rPsDef1). The full-length coding sequence of PsDef1 has an open reading frame capable to encode a protein of 83 amino residues, including a signal peptide of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5,601.6 Da. We have employed pET system to express mature form of PsDef1 fussed to GST. As GST-PsDef1 fusion protein was not biologically active, we removed GST moiety from the mature defensin 1 peptide by proteolytic cleavage with Factor Xa. The resulting rPsDef1 protein exhibited strong antifungal activity against a panel of pathogenic fungi which is comparable to that of endogenous Scots pine defensin 1. In addition, rPsDef1 was used to produce specific polyclonal antibodies. Using generated antibodies, we found that the level of PsDef1 is significantly increased in Scots pine seedlings during germination and in their response to pathogenic infection with Heterobasidion annosum.

Keywords: Antimicrobial peptides; Scots pine defensin; Recombinant PsDef1; Antifungal activity; PsDef1 specific antibodies

A bacterial laccase from marine microbial metagenome exhibiting chloride tolerance and dye decolorization ability by Zemin Fang; Tongliang Li; Quan Wang; Xuecheng Zhang; Hui Peng; Wei Fang; Yuzhi Hong; Honghua Ge; Yazhong Xiao (pp. 1103-1110).

Laccases are blue multicopper oxidases with potential applications in environmental and industrial biotechnology. In this study, a new bacterial laccase gene of 1.32 kb was obtained from a marine microbial metagenome of the South China Sea by using a sequence screening strategy. The protein (named as Lac15) of 439 amino acids encoded by the gene contains three conserved Cu²⁺-binding domains, but shares less than 40% of sequence identities with all of the bacterial multicopper oxidases characterized. Lac15, recombinantly expressed in Escherichia coli, showed high activity towards syringaldazine at pH 6.5–9.0 with an optimum pH of 7.5 and with the highest activity occurring at 45 °C. Lac15 was stable at pH ranging from 5.5 to 9.0 and at temperatures from 15 to 45 °C. Distinguished from fungal laccases, the activity of Lac15 was enhanced twofold by chloride at concentrations lower than 700 mM, and kept the original level even at 1,000 mM chloride. Furthermore, Lac15 showed an ability to decolorize several industrial dyes of reactive azo class under alkalescent conditions. The properties of alkalescence-dependent activity, high chloride tolerance, and dye decolorization ability make the new laccase Lac15 an alternative for specific industrial applications.

Keywords: Bacterial laccase; Chloride tolerance; Dye decolorization; Marine microbial metagenome; Multicopper oxidase

Biocatalytic properties of a recombinant aldo-keto reductase with broad substrate spectrum and excellent stereoselectivity by Yan Ni; Chun-Xiu Li; Hong-Min Ma; Jie Zhang; Jian-He Xu (pp. 1111-1118).

In the screening of 11 E. coli strains overexpressing recombinant oxidoreductases from Bacillus sp. ECU0013, an NADPH-dependent aldo-keto reductase (YtbE) was identified with capability of producing chiral alcohols. The protein (YtbE) was overexpressed, purified to homogeneity, and characterized of biocatalytic properties. The purified enzyme exhibited the highest activity at 50°C and optimal pH at 6.5. YtbE served as a versatile reductase showing a broad substrate spectrum towards different aromatic ketones and keto esters. Furthermore, a variety of carbonyl substrates were asymmetrically reduced by the purified enzyme with an additionally coupled NADPH regeneration system. The reduction system exhibited excellent enantioselectivity (>99% ee) in the reduction of all the aromatic ketones and high to moderate enantioselectivity in the reduction of α- and β-keto esters. Among the ketones tested, ethyl 4,4,4-trifluoroacetoacetate was found to be reduced to ethyl (R)-4,4,4-trifluoro-3-hydroxy butanoate, an important pharmaceutical intermediate, in excellent optical purity. To the best of our knowledge, this is the first report of ytbE gene-encoding recombinant aldo-keto reductase from Bacillus sp. used as biocatalyst for stereoselective reduction of carbonyl compounds. This study provides a useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers.

Keywords: YtbE; Recombinant aldo-keto reductase; Bacillus sp.; Asymmetric reduction; Chiral alcohols

Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose–xylose mixtures by Xiao-Jun Ji; Zhi-Kui Nie; He Huang; Lu-Jing Ren; Chao Peng; Ping-Kai Ouyang (pp. 1119-1125).

Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.

Keywords: 2,3-Butanediol; Klebsiella oxytoca ; Glucose; Xylose; Carbon catabolite repression; Mutant cAMP receptor protein

Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes by Taicheng Zhu; Meijin Guo; Yingping Zhuang; Ju Chu; Siliang Zhang (pp. 1127-1135).

Increased copy number of foreign gene can result in the alteration of normal metabolism in Pichia pastoris. To better understand the effect of foreign gene dosage on the cellular physiology of P. pastoris cells, comparative transcriptional analysis was performed among three P. pastoris strains carrying 0, 6, and 18 copies of porcine insulin precursor (PIP) expression cassettes, respectively. mRNA levels of 13 selected genes involved in methanol metabolic pathway, central metabolic pathway, protein folding, and oxidative stress were determined by real-time PCR. Results showed that enhanced PIP copy number resulted in an increase in PIP mRNA and also in folding stress on the yeast cells’ endoplasmic reticulum. The metabolism of 6-copy P. pastoris strain was not significantly changed as compared to 0-copy strain (control). In contrast, physiology of 18-copy strain was remarkably affected, characterized by the upregulation of antioxidative genes and readjusted expression level of methanol metabolic pathway genes. These data suggested that high copy P. pastoris strain might be suffering from protein folding-related oxidative stress and insufficient supply of carbon and energy sources.

Keywords: Gene dosage; Pichia pastoris ; Methanol metabolism; Cell physiology; mRNA quantitative analysis

Biosynthesis of zeaxanthin in recombinant Pseudomonas putida by Holger Beuttler; Jana Hoffmann; Marcel Jeske; Bernhard Hauer; Rolf D. Schmid; Josef Altenbuchner; Vlada B. Urlacher (pp. 1137-1147).

Pseudomonas putida KT2440 strain was investigated for biosynthesis of the valuable xanthophyll zeaxanthin. A new plasmid was constructed harboring five carotenogenic genes from Pantoea ananatis and three genes from Escherichia coli under control of an l-rhamnose-inducible promoter. Pseudomonas putida KT2440 wild type hardly tolerated the plasmids for carotenoid production. Mating experiments with E. coli S17-1 strains revealed that the carotenoid products are toxic to the Pseudomonas putida cells. Several carotenoid-tolerant transposon mutants could be isolated, and different gene targets for relief of carotenoid toxicity were identified. After optimization of cultivation conditions and product processing, 51 mg/l zeaxanthin could be produced, corresponding to a product yield of 7 mg zeaxanthin per gram cell dry weight. The effect of various additives on production of hydrophobic zeaxanthin was investigated as well. Particularly, the addition of lecithin during cell cultivation increased volumetric productivity of Pseudomonas putida by a factor of 4.7 (51 mg/l vs. 239 mg/l).

Keywords: Pseudomonas putida ; Zeaxanthin; Biosynthesis

Proteomic analysis of the GlnR-mediated response to nitrogen limitation in Streptomyces coelicolor M145 by Yvonne Tiffert; Mirita Franz-Wachtel; Claudia Fladerer; Alfred Nordheim; Jens Reuther; Wolfgang Wohlleben; Yvonne Mast (pp. 1149-1159).

GlnR is the global regulator of nitrogen assimilation in Streptomyces coelicolor M145 and other actinobacteria. Two-dimensional polyacrylamide gel electrophoresis analyses were performed to identify new GlnR target genes by proteomic comparison of wild-type S. coelicolor M145 and a ΔglnR mutant. Fifty proteins were found to be differentially regulated between S. coelicolor M145 and the ΔglnR mutant. These spots were identified by nanoHPLC–ESI-MS/MS and classified according to their cellular role. Most of the identified proteins are involved in amino acid biosynthesis and in carbon metabolism, demonstrating that the role of GlnR is not restricted to nitrogen metabolism. Thus, GlnR is supposed to play an important role in the global metabolic control of S. coelicolor M145.

Keywords: Actinomycetes; Ammonium assimilation; Glutamine synthetase; OmpR; Response regulator

Mechanisms of Bacillus cereus biofilm formation: an investigation of the physicochemical characteristics of cell surfaces and extracellular proteins by Esther Karunakaran; Catherine A. Biggs (pp. 1161-1175).

Microbial biofilms contribute to biofouling in a wide range of processes from medical implants to processed food. The extracellular polymeric substances (EPS) are implicated in imparting biofilms with structural stability and resistance to cleaning products. Still, very little is known about the structural role of the EPS in Gram-positive systems. Here, we have compared the cell surface and EPS of surface-attached (biofilm) and free-floating (planktonic) cells of Bacillus cereus, an organism routinely isolated from within biofilms on different surfaces. Our results indicate that the surface properties of cells change during biofilm formation and that the EPS proteins function as non-specific adhesions during biofilm formation. The physicochemical traits of the cell surface and the EPS proteins give us an insight into the forces that drive biofilm formation and maintenance in B. cereus.

Keywords: Bacillus cereus ; Biofilm; Cell surface; EPS; Proteins

Heterologous expression of Anabaena sp. PCC7120 cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium (Ensifer) meliloti 1021 by Yasser Abd-El-Karem; Tanja Elbers; Rudolf Reichelt; Alexander Steinbüchel (pp. 1177-1192).

Sinorhizobium meliloti infects leguminous plants resulting in a nitrogen-fixing symbiosis. Free living cells accumulate poly(3-hydroxybutyrate) (PHB) as carbon and energy source under imbalanced growth conditions. The cphA1 ₇₁₂₀ gene encoding a cyanophycin (CGP) synthetase of Anabaena sp. PCC7120 in plasmids pVLT31::cphA1 ₇₁₂₀ and pBBR1MCS-3::cphA1 ₇₁₂₀ was expressed in the wild-type S. meliloti 1021 and in a phbC-negative mutant generated in this study. Expression of cphA1 ₇₁₂₀ and accumulation of CGP in cells were studied in various media. Yeast mannitol broth (YMB) and pBBR1MCS-3::cphA1 ₇₁₂₀ yielded the highest CGP contents in both S. meliloti 1021 strains. Supplying the YMB medium with isopropyl-β-D-thiogalactopyranoside, aspartic acid, and arginine enhanced CGP contents about 2.5- and 2.8-fold in S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀), respectively. Varying the nitrogen-to-carbon ratio in the medium enhanced the CGP content further to 43.8% (w/w) of cell dry weight (CDW) in recombinant cells of S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀). Cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) accumulated CGP up to 39.6% in addition to 12.1% PHB (w/w, of CDW). CGP from the S. meliloti strains consisted of equimolar amounts of aspartic acid and arginine and contained no other amino acids even if the medium was supplemented with glutamic acid, citrulline, ornithine, or lysine. CGP isolated from cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀) exhibited average molecular weights between 20 and 25 kDa, whereas CGP isolated from Escherichia coli S17-1 (pBBR1MCS-3::cphA1 ₇₁₂₀) exhibited average molecular weight between 22 and 30 kDa. Co-expression of cyanophycinase from Anabaena sp. PCC7120 encoded by cphB1 ₇₁₂₀ in cphA1 ₇₁₂₀-positive E. coli S17-1, S. meliloti 1021, and its phbC-negative mutant gave cyanophycinase activities in crude extracts, and no CGP granules occurred. A higher PHB content in S. meliloti 1021 (pBBR1MCS-3::cphB1 ₇₁₂₀::cphA1 ₇₁₂₀) in comparison to the control indicated that the cells used CGP degradation product (β-aspartate-arginine dipeptide) to fuel PHB biosynthesis.

Keywords: Cyanophycin; Sinorhizobium meliloti ; Cyanophycinase; Cyanophycin synthetase; Poly-3-hydroxybutyrate

Comparison of the secondary metabolites in Penicillium chrysogenum between pilot and industrial penicillin G fermentations by Ying-Xiu Cao; Bin Qiao; Hua Lu; Yao Chen; Ying-Jin Yuan (pp. 1193-1202).

The disparity of secondary metabolites in Penicillium chrysogenum between two scales of penicillin G fermentation (50 L as pilot process and 150,000 L as industrial one) was investigated by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry. In industrial process, the pools of intracellular L-α-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) and isopenicillin N (IPN) were remarkably less than that in the pilot one, which indicated that the productivity of penicillin G might be higher in the large scale of fermentation. This conclusion was supported by the higher intracellular penicillin G concentration as well as its higher yield per unit biomass in industrial cultivation. The different changing tendencies of IPN, 6-aminopenicillanic acid and 6-oxopiperide-2-carboxylic acid between two processes also suggested the same conclusion. The higher content of intracellular LLD-ACV in pilot process lead to a similarly higher concentration of bis-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, which had an inhibitory effect on ACV synthetase and also subdued the activity of IPN synthetase. The interconversion of secondary metabolites and the influence they put on enzymes would intensify the discrepancy between two fermentations more largely. These findings provided new insight into the changes and regulation of secondary metabolites in P. chrysogenum under different fermentation sizes.

Keywords: Penicillium chrysogenum ; Penicillin fermentation; Scale-up; Secondary metabolites; Q-TOF-MS

Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples by José L. Alonso; Inmaculada Amorós; Irene Cañigral (pp. 1203-1211).

Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the β-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower C _t values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier C _t values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.

Keywords: Cryptosporidium ; Giardia ; LightCycler; LNA probe; qPCR; Sewage; TaqMan

Development of an autonomously replicating linear vector of the yeast Cryptococcus humicola by using telomere-like sequence repeats by Shouji Takahashi; Yumie Nakajima; Tomomi Imaizumi; Yudai Furuta; Yuichi Ohshiro; Katsumasa Abe; Ryo-hei Yamada; Yoshio Kera (pp. 1213-1221).

The yeast Cryptococcus humicola has several attractive properties for practical applications such as in bioremediation and as a source of industrially useful enzymes and compounds. We have developed an autonomously replicating vector of C. humicola to improve its properties. We initially tried to isolate an autonomously replicating sequence (ARS) from genomic DNA by transformation using a genomic DNA library. We obtained a candidate plasmid vector harboring an ARS that gave high transformation efficiency. Southern blot analysis of transformants revealed the autonomous replication of the introduced vector in some transformants. However, the vector was not only variously altered in length but also linearized. PCR analysis indicated that a telomere-like sequence repeat (TTAGGGGG) n was added to the termini of linearized vector. Thus, we constructed an autonomously replicating linear vector having ten repeats of the telomere-like sequence at both ends. The vector transformed the yeast cells with high transformation efficiency (3230 CFU/μg of DNA), which was approximately 25-fold higher than that of a control vector lacking the repeats, and was autonomously replicated at a roughly constant size. The copy number was estimated to be less than one copy, and Ura⁺ mitotic stability varied widely among the transformants and was related to plasmid segregation efficiency.

Keywords: Cryptococcus humicola ; Autonomously replicating vector; Transformation; Telomere; Yeast

Decolourization of synthetic wastewater containing azo dyes by immobilized Phanerochaete chrysosporium in a continuously operated RBC reactor by Kannan Pakshirajan; Auta Sivasankar; Naresh Kumar Sahoo (pp. 1223-1232).

A laboratory-scale rotating biological contactor (RBC) reactor with immobilized fungal biomass of Phanerochaete chrysosporium was investigated for its performance in decolourizing synthetic wastewater containing single or mixture of azo dyes, Direct Red-80 (DR-80) and Mordant Blue-9 (MB-9). Decolourization efficiency in the continuously operated bioreactor was studied by varying dye inlet concentration and disc rotation speed at two different wastewater hydraulic retention times (HRTs), i.e. 24and 48 h. Results from the single dye-containing experiments showed that the system could completely decolourize the wastewater for a maximum inlet dye concentration within the range 25–200 mg L⁻¹ and 48 h HRT in the reactor; for an inlet dye concentration above 200 mg L⁻¹, the decolourization efficiency slightly reduced up to 85% for the same HRT. However, wastewater containing DR-80 was found to be decolourized more efficiently compared to that containing MB-9. Further, the effect of increase in the disc rotation speed from 2 to 6 rpm in the study revealed no large differences in the decolourization efficiencies of the wastewaters. Similar results were obtained with wastewater containing the dyes together at various concentration combinations as per the two-level factorial design of experiments. Enzyme activities of lignin peroxidase and manganese peroxidase by the fungus were also analysed in the study, and the results indicated that while DR-80 showed a large negative effect on both the enzymes, MB-9 affected mainly the MnP activity by the fungus.

Keywords: Azo dyes; Direct Red-80; Mordant Blue-9; Phanerochaete chrysosporium ; Rotating biological contactor reactor; Wastewater decolourization

Diversity and abundance of anammox bacterial community in the deep-ocean surface sediment from equatorial Pacific by Yi-Guo Hong; Bo Yin; Tian-Ling Zheng (pp. 1233-1241).

The community structure and diversity of anaerobic ammonium oxidation (anammox) bacteria in the surface sediments of equatorial Pacific were investigated by phylogenic analysis of 16S rRNA and hydrazine oxidoreductase (hzo) genes and PCoA (principal coordinates analysis) statistical analysis. Results indicated that 16S rRNA and hzo sequences in the P2 (off the center of western Pacific warm pool) and P3 (in the eastern equatorial Pacific) sites all belong to the Candidatus “Scalindua”, the dominate anammox bacteria in the low-temperature marine environment proved by previous studies. However, in the P1 site (in center of warm pool of western Pacific), large part of 16S rRNA gene sequences formed a separated cluster. Meanwhile, hzo gene sequences from P1 sediment also grouped into a single cluster. PCoA analysis demonstrated that the anammox community structure in the P1 has significant geographical distributional difference from that of P2, P3, and other marine environments based on 16S rRNA and hzo genes. The abundances of anammox bacteria in surface sediments of equatorial Pacific were quantified by q-PCR analysis of hzo genes, which ranged from 3.98 × 10³ to 1.17 × 10⁴ copies g⁻¹ dry sediments. These results suggested that a special anammox bacteria phylotypes exist in the surface sediment of the western Pacific warm pool, which adapted to the specific habitat and maybe involved in the nitrogen loss process from the fixed inventory in the habitat.

Keywords: Anammox; 16S rRNA gene; hzo gene; Surface sediment; Western pacific warm pool

Spatial distribution and abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in mangrove sediments by Meng Li; Huiluo Cao; Yiguo Hong; Ji-Dong Gu (pp. 1243-1254).

We investigated the diversity, spatial distribution, and abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in sediment samples of different depths collected from a transect with different distances to mangrove forest in the territories of Hong Kong. Both the archaeal and bacterial amoA genes (encoding ammonia monooxygenase subunit A) from all samples supported distinct phylogenetic groups, indicating the presences of niche-specific AOA and AOB in mangrove sediments. The higher AOB abundances than AOA in mangrove sediments, especially in the vicinity of the mangrove trees, might indicate the more important role of AOB on nitrification. The spatial distribution showed that AOA had higher diversity and abundance in the surface layer sediments near the mangrove trees (0 and 10 m) but lower away from the mangrove trees (1,000 m), and communities of AOA could be clustered into surface and bottom sediment layer groups. In contrast, AOB showed a reverse distributed pattern, and its communities were grouped by the distances between sites and mangrove trees, indicating mangrove trees might have different influences on AOA and AOB community structures. Furthermore, the strong correlations among archaeal and bacterial amoA gene abundances and their ratio with NH ₄ ⁺ , salinity, and pH of sediments indicated that these environmental factors have strong influences on AOA and AOB distributions in mangrove sediments. In addition, AOA diversity and abundances were significantly correlated with hzo gene abundances, which encodes the key enzyme for transformation of hydrazine into N₂ in anaerobic ammonium-oxidizing (anammox) bacteria, indicating AOA and anammox bacteria may interact with each other or they are influenced by the same controlling factors, such as NH ₄ ⁺ . The results provide a better understanding on using mangrove wetlands as biological treatment systems for removal of nutrients.

Keywords: Ammonia-oxidizing archaea (AOA); Ammonia-oxidizing bacteria (AOB); Community structures; Mangrove sediments; Anammox; hzo gene; Abundance; Diversity

Ethanol production from biodiesel-derived crude glycerol by newly isolated Kluyvera cryocrescens by Won Jae Choi; Maria Regina Hartono; Weng Heng Chan; Suan Siong Yeo (pp. 1255-1264).

The rapidly expanding market for biodiesel has increased the supply and reduced the cost of glycerol, making it an attractive sustainable feed stock for the fuel and chemical industry. Glycerol-based biorefinery is the microbial fermentation of crude glycerol to produce fuels and chemicals. A major challenge is to obtain microbes tolerant to inhibitors such as salts and organic solvents present in crude glycerol. Microbial screening was attempted to isolate novel strain capable of growing on crude glycerol as a sole carbon source. The newly isolated bacteria, identified as nonpathogenic Kluyvera cryocrescens S26 could convert biodiesel-derived crude glycerol to ethanol with high yield and productivity. The supplementation of nutrients such as yeast extract resulted in distinguished enhancement in cell growth as well as ethanol productivity under anaerobic condition. When glycerol fermentation is performed under microaerobic condition, there is also a remarkable improvement in cell growth, ethanol productivity and yield, compared with those under strict anaerobic condition. In batch fermentation under microaerobic condition, K. cryocrescens S26 produced 27 g/l of ethanol from crude glycerol with high molar yield of 80% and productivity of 0.61 g/l/h.

Keywords: Crude glycerol; Ethanol; Fermentation; Kluyvera cryocrescens

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Applied Microbiology and Biotechnology (v.89, #4)

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Applied Microbiology and Biotechnology (v.89, #4)