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Applied Microbiology and Biotechnology (v.97, #3)

Tubular membrane bioreactors for biotechnological processes by Christoph Wolff; Sascha Beutel; Thomas Scheper (pp. 929-937).

This article is an overview of bioreactors using tubular membranes such as hollow fibers or ceramic capillaries for cultivation processes. This diverse group of bioreactor is described here in regard to the membrane materials used, operational modes, and configurations. The typical advantages of this kind of system such as environments with low shear stress together with high cell densities and also disadvantages like poor oxygen supply are summed up. As the usage of tubular membrane bioreactors is not restricted to a certain organism, a brief overview of various applications covering nearly all types of cells from prokaryotic to eukaryotic cells is also given here.

Keywords: Tubular membranes; Bioreactor; Cultivation; Immobilization

Environmental responses and the control of iron homeostasis in fungal systems by Paulo Canessa; Luis F. Larrondo (pp. 939-955).

Organisms need to actively respond to changes in the environment and, particularly under diverse conditions, they ought to ensure access to nutrients. Among micronutrients, iron is a key component of several enzymes and participates in a variety of cellular processes. Iron deprivation therefore poses a serious challenge to both unicellular and multicellular individuals. Nevertheless, excess of this metal is toxic, compromising cell function and viability. Thus, it is not surprising that organisms have evolved sophisticated mechanisms to tightly regulate cellular iron levels. In the last decade, major advances have been achieved in the molecular understanding of how fungi respond to changing iron concentrations. Moreover, this metal has been recognized as an important element impacting pathogenic and saprophytic fungal lifestyles. An interconnected transcriptional negative feedback loop has been described as central in the regulation of genes encoding for iron uptake and utilization components in fungi. The observation that light, oxygen, or nutrients can also impact the expression of some of these elements suggests that additional environmental inputs—besides iron levels—may as well modulate the machinery underpinning iron homeostasis. This review highlights some of the latest findings associated with iron-regulated processes in fungi and revisits the increasing transcriptional complexity involved in the control of this metal homeostasis. In addition, we present the first in silico evidence of genes encoding for putative ferritins in zygomycetes and chytrids, as well as other ferritin-like sequences widespread among fungi, which raises interesting questions relative to iron storage in this particular group of organisms.

Keywords: Iron; Fenton reaction; Light; Ferritin; Ferroxidases

Current knowledge and perspectives on biofilm formation: the case of Listeria monocytogenes by Eliane Pereira da Silva; Elaine Cristina Pereira De Martinis (pp. 957-968).

Listeriosis is a rare, serious, and mainly food-borne infection caused by the bacterium Listeria monocytogenes. This food-borne infection primarily affects pregnant women and immunologically compromised individuals. L. monocytogenes is recognized as a problem for the food industry, mainly due to its environmental persistence, attributed in part to its ability to form biofilms. Biofilms are microbial communities adhered to biotic or abiotic surfaces coated by self-produced extracellular polymers. These structures confer protection to bacterial cells and decrease the efficiency of cleaning and disinfection procedures. This article presents a brief review of current perspectives on the formation of biofilms, with emphasis on L. monocytogenes, highlighting the importance of cell-to-cell communication and structural composition of the microbial communities. The techniques currently used to study biofilms and the need to develop new strategies for the prevention and control of biofilm-forming pathogens are also discussed.

Keywords: Biofilms; Listeria monocytogenes ; Listeriosis; In vitro techniques; Prevention and control

Antimicrobials, drug discovery, and genome mining by R. J. Scheffler; S. Colmer; H. Tynan; A. L. Demain; V. P. Gullo (pp. 969-978).

Over the years, antibiotics have provided an effective treatment for a number of microbial diseases. However recently, there has been an increase in resistant microorganisms that have adapted to our current antibiotics. One of the most dangerous pathogens is methicillin-resistant Staphylococcus aureus (MRSA). With the rise in the cases of MRSA and other resistant pathogens such as vancomycin-resistant Staphylococcus aureus, the need for new antibiotics increases every day. Many challenges face the discovery and development of new antibiotics, making it difficult for these new drugs to reach the market, especially since many of the pharmaceutical companies have stopped searching for antibiotics. With the advent of genome sequencing, new antibiotics are being found by the techniques of genome mining, offering hope for the future.

Keywords: Genome mining; Discovery; MRSA; Targets; Antibiotics

What do we know about the yeast strains from the Brazilian fuel ethanol industry? by Bianca Eli Della-Bianca; Thiago Olitta Basso; Boris Ugarte Stambuk; Luiz Carlos Basso; Andreas Karoly Gombert (pp. 979-991).

The production of fuel ethanol from sugarcane-based raw materials in Brazil is a successful example of a large-scale bioprocess that delivers an advanced biofuel at competitive prices and low environmental impact. Two to three fed-batch fermentations per day, with acid treatment of the yeast cream between consecutive cycles, during 6–8 months of uninterrupted production in a nonaseptic environment are some of the features that make the Brazilian process quite peculiar. Along the past decades, some wild Saccharomyces cerevisiae strains were isolated, identified, characterized, and eventually, reintroduced into the process, enabling us to build up knowledge on these organisms. This information, combined with physiological studies in the laboratory and, more recently, genome sequencing data, has allowed us to start clarifying why and how these strains behave differently from the better known laboratory, wine, beer, and baker's strains. All these issues are covered in this minireview, which also presents a brief discussion on future directions in the field and on the perspectives of introducing genetically modified strains in this industrial process.

Keywords: Yeast; Fuel ethanol; Saccharomyces cerevisiae ; Industrial microbiology; Alcoholic fermentation

Radiation-resistant extremophiles and their potential in biotechnology and therapeutics by Prashant Gabani; Om V. Singh (pp. 993-1004).

Extremophiles are organisms able to thrive in extreme environmental conditions. Microorganisms with the ability to survive high doses of radiation are known as radioresistant or radiation-resistant extremophiles. Excessive or intense exposure to radiation (i.e., gamma rays, X-rays, and particularly UV radiation) can induce a variety of mutagenic and cytotoxic DNA lesions, which can lead to different forms of cancer. However, some populations of microorganisms thrive under different types of radiation due to defensive mechanisms provided by primary and secondary metabolic products, i.e., extremolytes and extremozymes. Extremolytes (including scytonemin, mycosporine-like amino acids, shinorine, porphyra-334, palythine, biopterin, and phlorotannin, among others) are able to absorb a wide spectrum of radiation while protecting the organism’s DNA from being damaged. The possible commercial applications of extremolytes include anticancer drugs, antioxidants, cell-cycle-blocking agents, and sunscreens, among others. This article aims to review the strategies by which microorganisms thrive in extreme radiation environments and discuss their potential uses in biotechnology and the therapeutic industry. The major challenges that lie ahead are also discussed.

Keywords: Radiation; Microorganisms; Extremophiles; Extremolytes; Extremozymes

Biosurfactants in agriculture by Dhara P. Sachdev; Swaranjit S. Cameotra (pp. 1005-1016).

Agricultural productivity to meet growing demands of human population is a matter of great concern for all countries. Use of green compounds to achieve the sustainable agriculture is the present necessity. This review highlights the enormous use of harsh surfactants in agricultural soil and agrochemical industries. Biosurfactants which are reported to be produced by bacteria, yeasts, and fungi can serve as green surfactants. Biosurfactants are considered to be less toxic and eco-friendly and thus several types of biosurfactants have the potential to be commercially produced for extensive applications in pharmaceutical, cosmetics, and food industries. The biosurfactants synthesized by environmental isolates also has promising role in the agricultural industry. Many rhizosphere and plant associated microbes produce biosurfactant; these biomolecules play vital role in motility, signaling, and biofilm formation, indicating that biosurfactant governs plant–microbe interaction. In agriculture, biosurfactants can be used for plant pathogen elimination and for increasing the bioavailability of nutrient for beneficial plant associated microbes. Biosurfactants can widely be applied for improving the agricultural soil quality by soil remediation. These biomolecules can replace the harsh surfactant presently being used in million dollar pesticide industries. Thus, exploring biosurfactants from environmental isolates for investigating their potential role in plant growth promotion and other related agricultural applications warrants details research. Conventional methods are followed for screening the microbial population for production of biosurfactant. However, molecular methods are fewer in reaching biosurfactants from diverse microbial population and there is need to explore novel biosurfactant from uncultured microbes in soil biosphere by using advanced methodologies like functional metagenomics.

Keywords: Biosurfactants; Sustainable agriculture; Pesticide; Plant pathogen; Remediation; Metagenomics

Application of hairy roots for phytoremediation: what makes them an interesting tool for this purpose? by Elizabeth Agostini; Melina A. Talano; Paola S. González; Ana L. Wevar Oller; María I. Medina (pp. 1017-1030).

In recent years, hairy roots (HRs) have been successfully used as research tools for screening the potentialities of different plant species to tolerate, accumulate, and/or remove environmental pollutants, such as PCBs, TNT, pharmaceuticals, textile dyes, phenolics, heavy metals, and radionuclides. This is in part due to several advantages of this plant model system and the fact that roots have evolved specific mechanisms to deal with pollutants because they are the first organs to have contact with them. In addition, by using HRs some metabolic pathways and enzymatic catalyzed reactions involved in pollutants detoxification can be elucidated as well as the mechanisms of uptake, transformation, conjugation, and compartmentation of pollutants in vacuoles and/or cell walls, which are important detoxification sites in plants. Plant roots also stimulate the degradation of contaminants by the release of root exudates and oxido-reductive enzymes, such as peroxidases (Px) and laccases, that are associated with the removal of some organic pollutants. HRs are also considered good alternatives as enzyme sources for remediation purposes. Furthermore, application of genetic engineering methods and development of microbe-assisted phytoremediation are feasible strategies to enhance plant capabilities to tolerate, accumulate, and/or metabolize pollutants and, hence, to create or find an appropriate plant system for environmental cleanup. The present review highlights current knowledge, recent progress, areas which need to be explored, and future perspectives related to the application and improvement of the efficiency of HRs for phytoremediation research.

Keywords: Hairy roots; Phytoremediation; Organic pollutant; Inorganic pollutant; Phenol

Glycerol-based sterilization bioindicator system from Bacillus atrophaeus: development, performance evaluation, and cost analysis by Sandra R. B. R. Sella; Patricia Milla Gouvea; Vanessa F. Gomes; Luciana P. S. Vandenberghe; João Carlos Minozzo; Carlos Ricardo Soccol (pp. 1031-1042).

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D _160 °C value of 6.6 ± 0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10⁸ CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.

Keywords: Glycerol; Bacillus atrophaeus ; Sterilization bioindicator; Spores; Recovery medium; Solid-state fermentation

Enhanced chemical and biological activities of a newly biosynthesized eugenol glycoconjugate, eugenol α-d-glucopyranoside by Peng Zhang; Erli Zhang; Min Xiao; Chang Chen; Weijian Xu (pp. 1043-1050).

Eugenol, the essential component (over 90 %) of clove oil from Eugenia caryophyllata Thunb. (Myrtaceae), is a phenolic compound well known for its versatile pharmacological actions, including analgesic, local anesthetic, anti-inflammatory, antimicrobial, antitumor, and hair-growing effects. However, the application of eugenol is greatly limited mainly because of its unwanted physicochemical properties, such as low solubility, liability to sublimation, and pungent odor. Since glycosylation has been suggested to improve the physicochemical and biological properties of the parental compound, we have previously developed a novel and efficient way to biosynthesize highly purified eugenol α-D-glucopyranoside (α-EG). In light of the widely acknowledged importance of pure eugenol and the potential superiority of the glycosylation, it is crucial to further explore and compare the physicochemical and biological properties of these two phenolic compounds. In this study, we demonstrate that glucosylation is a promising method for modification of phenolic compound, and that α-EG is superior over its parent eugenol, in all of the tested aspects, including physicochemical properties, antioxidation activity, and antimicrobial and antitumor activities. These results strongly suggest that α-EG, as a novel prodrug, may serve as a useful probe and potential therapeutic drug in both fundamental research and clinical application in the coming future.

Keywords: Phenolic compound; Glucosylation; Eugenol α-d-glucopyranoside; Biological activity

Pathway of cytotoxicity induced by folic acid modified selenium nanoparticles in MCF-7 cells by Jiang Pi; Hua Jin; RuiYing Liu; Bing Song; Qing Wu; Li Liu; JinHuan Jiang; Fen Yang; HuaiHong Cai; Jiye Cai (pp. 1051-1062).

Selenium nanoparticles (Se NPs) have been recognized as promising materials for biomedical applications. To prepare Se NPs which contained cancer targeting methods and to clarify the cellular localization and cytotoxicity mechanisms of these Se NPs against cancer cells, folic acid protected/modified selenium nanoparticles (FA–Se NPs) were first prepared by a one-step method. Some morphologic and spectroscopic methods were obtained to prove the successfully formation of FA–Se NPs while free folate competitive inhibition assay, microscope, and several biological methods were used to determine the in vitro uptake, subcellular localization, and cytotoxicity mechanism of FA–Se NPs in MCF-7 cells. The results indicated that the 70-nm FA–Se NPs were internalized by MCF-7 cells through folate receptor-mediated endocytosis and targeted to mitochondria located regions through endocytic vesicles transporting. Then, the FA–Se NPs entered into mitochondria; triggered the mitochondria-dependent apoptosis of MCF-7 cells which involved oxidative stress, Ca²+ stress changes, and mitochondrial dysfunction; and finally caused the damage of mitochondria. FA–Se NPs released from broken mitochondria were transported into nucleus and further into nucleolus which then induced MCF-7 cell cycle arrest. In addition, FA–Se NPs could induce cytoskeleton disorganization and induce MCF-7 cell membrane morphology alterations. These results collectively suggested that FA–Se NPs could be served as potential therapeutic agents and organelle-targeted drug carriers in cancer therapy.

Keywords: Apoptosis; Endocytosis; Folate; MCF-7 cells; Selenium nanoparticles; Subcellular localization

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor by Bo Sun; XiangHui Yu; Wei Kong; Shiyang Sun; Ping Yang; Changlin Zhu; Haihong Zhang; Yongge Wu; Yan Chen; Yuhua Shi; Xizhen Zhang; Chunlai Jiang (pp. 1063-1070).

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0 × 10⁹ to 3.2 × 10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8 × 10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.

Keywords: Influenza virus; MDCK cells; Vaccine production; Bioreactor

Efficient production of Japanese encephalitis virus-like particles by recombinant lepidopteran insect cells by Hideki Yamaji; Masataka Nakamura; Miwa Kuwahara; Yusuke Takahashi; Tomohisa Katsuda; Eiji Konishi (pp. 1071-1079).

The production of Japanese encephalitis (JE) virus-like particles (VLPs) in stably transformed lepidopteran insect cells was investigated. The DNA fragment encoding the JE virus (JEV) prM signal peptide, the precursor (prM) of the viral membrane protein (M), and the envelope glycoprotein (E) was cloned into the plasmid vector pIHAbla. The pIHAbla contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression, together with a blasticidin resistance gene for use as a selectable marker. DNA encoding a form of prM with a pr/M cleavage site mutation was used to suppress the cell-fusion activity of VLPs. After transfection with the resultant plasmid, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were incubated with blasticidin, and cells resistant to the antibiotic were obtained. Western blot analysis and enzyme-linked immunosorbent assay of a culture supernatant showed that transfected High Five cells secreted an E antigen equivalent to the authentic JEV E. Sucrose density-gradient sedimentation analysis of the culture supernatant from recombinant High Five cells indicated that secreted E antigen molecules were produced in a particulate form. VLPs recovered from the supernatant successfully induced neutralizing antibodies in mice, particularly when adsorbed to alum adjuvant. High yields (≈30 μg/ml) of E antigen were achieved in shake-flask cultures. These results indicate that recombinant insect cells may offer a novel approach for efficient VLP production.

Keywords: Insect cell culture; Recombinant protein production; Virus-like particles; Japanese encephalitis virus; High Five cells

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO₂ nanoparticles by Saurabh Sudha Dhiman; Dayanand Kalyani; Sujit Sadashiv Jagtap; Jung-Rim Haw; Yun Chan Kang; Jung-Kul Lee (pp. 1081-1091).

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and β-glucosidase activities of 1,270, 146, 34, and 15 U mL⁻¹, respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k _cat/K _m = 1,440 mg mL⁻¹ s⁻¹) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.

Keywords: Denaturation constant; Enthalpy; Immobilization; SiO₂ ; Xylanase; Xylooligosaccharide

Growth and characterization of Escherichia coli DH5α biofilm on concrete surfaces as a protective layer against microbiologically influenced concrete deterioration (MICD) by Sahar Soleimani; Banu Ormeci; O. Burkan Isgor (pp. 1093-1102).

Biofilms of selected bacteria strains were previously used on metal coupons as a protective layer against microbiologically influenced corrosion of metals. Unlike metal surfaces, concrete surfaces present a hostile environment for growing a protective biofilm. The main objective of this research was to investigate whether a beneficial biofilm can be successfully grown on mortar surfaces. Escherichia coli DH5α biofilm was grown on mortar surfaces for 8 days, and the structure and characteristics of the biofilm were studied using advanced microscopy techniques such as scanning electron microscopy and confocal laser scanning microscopy in combination with fluorescence in situ hybridization, live/dead, extracellular polymer staining, ATP analysis, and membrane filtration. A biofilm layer with a varying thickness of 20–40 μm was observed on the mortar surface. The distribution of live and dead bacteria and extracellular polymers varied with depth. The density of the live population near the mortar surface was the lowest. The bacteria reached their highest density at three fourths of the biofilm depth and then decreased again near the biofilm–liquid interface. Overall, the results indicated a healthy biofilm growth in the chosen growth period of 8 days, and it is expected that longer growth periods would lead to formation of a more resistant biofilm with more coverage of mortar surfaces.

Keywords: E. coli ; Biofilm; Microbiologically influenced concrete deterioration (MICD); Sulfur-oxidizing bacteria (SOB)

Production of sophorolipids with enhanced volumetric productivity by means of high cell density fermentation by Renjun Gao; Mia Falkeborg; Xuebing Xu; Zheng Guo (pp. 1103-1111).

To achieve high time–space efficiency for sophorolipid production with yeast Candida bombicola, a strategy of high cell density fermentation was employed. The approach consisted of two sequential stages: (1) the optimization of the carbon source and the nutrient concentration to achieve the maximal cell density and (2) the computer-aided adjustment of physical parameters and the controlled feeding of substrates for enhanced volumetric productivity. Both stages have been successfully implemented in a 10-L fermenter, where up to 80 g dry cell weight/L was obtained and a remarkably high volumetric productivity (> 200 g isolated sophorolipids/L/day) was achieved. Both the biomass and volumetric productivity were markedly higher than previously reported. Specifically, the high productivity of sophorolipids could be attained on a very short time scale (24 h), highlighting the industrial potential of the platform developed in this work.

Keywords: Sophorolipids; Candida bombicola ; Volumetric productivity; High cell density fermentation; Biosurfactants

High-level expression, purification, and enzymatic characterization of truncated poly(vinyl alcohol) dehydrogenase in methylotrophic yeast Pichia pastoris by Dongxu Jia; Jianghua Li; Long Liu; Dongxu Zhang; Yu Yang; Guocheng Du; Jian Chen (pp. 1113-1120).

A 1,965-bp fragment encoding a poly(vinyl alcohol) dehydrogenase (PVADH) from Sphingopyxis sp. 113P3 was synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. The fragment was then amplified by polymerase chain reaction and inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the AOX1 promoter and α-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmid, designated as pPIC9K-PVADH, was linearized using SalI and transformed into P. pastoris GS115 by electroporation. The PVADH activity reached 55 U/mL in a shake flask and 902 U/mL in a 3-L bioreactor. Surprisingly, the sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and N-terminal sequencing indicated that the secreted PVADH was truncated, and it had only 548 amino acid residues (an 81-amino acid sequence from the secreted protein was cleaved). The optimum pH and temperature ranges for the truncated PVADH were 7.0–8.0 and 41–53 °C, respectively. The activation energy of the recombinant truncated PVADH was approximately 10.36 kcal/mol between 29 and 41 °C. Both Ca²⁺ and Mg²⁺ had stimulating effects on the activity of PVADH. With PVA1799 as the substrate, the truncated PVADH had a Michaelis constant (K _m) of 1.89 mg/mL and a maximum reaction rate (V _max) of 34.9 nmol/(min mg protein). To the best of our knowledge, this is the first report on the expression of PVADH in P. pastoris, and the achieved PVADH yield is the highest ever reported.

Keywords: Poly(vinyl alcohol) dehydrogenase; Pichia pastoris ; Expression; Characterization

Substrate specificity and gene expression of two Penicillium chrysogenum α-l-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54 by Tatsuji Sakamoto; Misako Inui; Kana Yasui; Sachiko Hosokawa; Hideshi Ihara (pp. 1121-1130).

We previously isolated two α-l-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an “Alpha-L-AF_C” domain in AFQ1 and “ArabFuran-catal” and “AbfB” domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-l-arabinofuranoside and polysaccharides with different specificities. ¹H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and l-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.

Keywords: α-l-Arabinofuranosidase; Glycoside hydrolase family 51; Glycoside hydrolase family 54; Expression; Penicillium chrysogenum

Substrate range and enantioselectivity of epoxidation reactions mediated by the ethene-oxidising Mycobacterium strain NBB4 by Samantha Cheung; Victoria McCarl; Andrew J. Holmes; Nicholas V. Coleman; Peter J. Rutledge (pp. 1131-1140).

Mycobacterium strain NBB4 is an ethene-oxidising micro-organism isolated from estuarine sediments. In pursuit of new systems for biocatalytic epoxidation, we report the capacity of strain NBB4 to convert a diverse range of alkene substrates to epoxides. A colorimetric assay based on 4-(4-nitrobenzyl)pyridine) has been developed to allow the rapid characterisation and quantification of biocatalytic epoxide synthesis. Using this assay, we have demonstrated that ethene-grown NBB4 cells epoxidise a wide range of alkenes, including terminal (propene, 1-butene, 1-hexene, 1-octene and 1-decene), cyclic (cyclopentene, cyclohexene), aromatic (styrene, indene) and functionalised substrates (allyl alcohol, dihydropyran and isoprene). Apparent specific activities have been determined and range from 2.5 to 12.0 nmol min⁻¹ per milligram of cell protein. The enantioselectivity of epoxidation by Mycobacterium strain NBB4 has been established using styrene as a test substrate; (R)-styrene oxide is produced in enantiomeric excesses greater than 95%. Thus, the ethene monooxygenase of Mycobacterium NBB4 has a broad substrate range and promising enantioselectivity, confirming its potential as a biocatalyst for alkene epoxidation.

Keywords: Biocatalysis; Epoxidation; Alkene; Mycobacterium ; Soluble di-iron monooxygenase; Non-heme iron; Oxidation

Role and fate of SP100 protein in response to Rep-dependent nonviral integration system by Yuan-Yuan Xue; Ran Wang; Yang-Bo Yue; Jing-Lun Xue; Jin-Zhong Chen (pp. 1141-1147).

Previously, we studied an AAVS1 site-specific non-viral integration system with a Rep-donor plasmid and a plasmid containing adeno-associated virus integration element. Our earlier study focused on the plasmid vector itself, but the cellular response to the system was still unknown. SP100 is a member of the promyelocytic leukemia nuclear bodies. It is involved in many cellular processes such as transcriptional regulation and the cellular intrinsic immune response against viral infection. In this study, we revealed that SP100 inhibited the Rep-dependent nonviral integration. Conversely, transient expression of Rep78 increased the degradation of SP100. This degradation was inhibited by treatment with MG132, an inhibitor of the ubiquitin proteasome. SP100 and Rep78 are both located in the nucleolus, which provides the spatial possibility for their interaction. Rep78 was coimmunoprecipitated with the enhanced green fluorescent protein (EGFP)–SP100 fusion protein but not EGFP, which verified the interaction between Rep78 and SP100. These results have enriched our knowledge about the cellular protein SP100 and Rep-dependent nonviral integration. It may lead to an improvement in the application of Rep-related transgene integration method and in the selection of target cells.

Keywords: SP100; Rep78; Integration; Degradation

Immobilization of glucoamylase onto polyaniline-grafted magnetic hydrogel via adsorption and adsorption/cross-linking by Gulay Bayramoglu; Begum Altintas; M. Yakup Arica (pp. 1149-1159).

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of “starch” dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7 mg/g from 2.0 mg/mL enzyme solution at pH 4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55 °C and pH 4.5 compared to free enzyme (50 °C and pH 5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase.

Keywords: Ferrogel; Film; Enzyme immobilization; Adsorption; Glucoamylase; Starch hydrolysis

Carboxylesterase 2 production and characterization in human cells: new insights into enzyme oligomerization and activity by Joana Lamego; Bárbara Cunha; Cristina Peixoto; Marcos F. Sousa; Paula M. Alves; Ana L. Simplício; Ana S. Coroadinha (pp. 1161-1173).

Carboxylesterase 2 (CES2), the main carboxylesterase expressed in human intestine, is an increasingly important enzyme in anti-cancer combined therapies for the treatment of different pathologies like colon adenocarcinoma and malignant glioma. The production of human recombinant CES2, in human embryonic kidney cells (HEK-293T cells) using serum-free media, is herein described. CES2 secretion to the media was achieved by the simple addition of an in-frame C-terminal 10× histidine tag (CES2-10xHis) without the need of addition of extra N-terminal signalling sequences or the mutation or deletion of the C-terminal HTEL motif responsible for retaining the protein in the lumen of endoplasmic reticulum. This secretion allowed a fourfold increase in CES2 production. The characterization of human recombinant CES2 showed that this protein exists in other active and inactive forms than the described 60 kDa monomer.

Keywords: Carboxylesterases; Human cells; Protein expression; Enzyme characterization

New insights into activation and substrate recognition of polyhydroxyalkanoate synthase from Ralstonia eutropha by Kazunori Ushimaru; Smith Sangiambut; Nicholas Thomson; Easan Sivaniah; Takeharu Tsuge (pp. 1175-1182).

The polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC_Re) shows a lag time for the start of its polymerization reaction, which complicates kinetic analysis of PhaC_Re. In this study, we found that the lag can be virtually eliminated by addition of 50 mg/L TritonX-100 detergent into the reaction mixture, as well as addition of 2.5 g/L Hecameg detergent as previously reported by Gerngross and Martin (Proc Natl Sci USA 92: 6279–6283, 1995). TritonX-100 is an effective lag eliminator working at much lower concentration than Hecameg. Kinetic analysis of PhaC_Re was conducted in the presence of TritonX-100, and PhaC_Re obeyed Michaelis–Menten kinetics for (R)-3-hydroxybutyryl-CoA substrate. In inhibitory assays using various compounds such as adenosine derivatives and CoA derivatives, CoA free acid showed competitive inhibition but other compounds including 3′-dephospho CoA had no inhibitory effect. Furthermore, PhaC_Re showed a considerably reduced reaction rate for 3′-dephospho (R)-3-hydroxybutyryl CoA substrate and did not follow typical Michaelis–Menten kinetics. These results suggest that the 3′-phosphate group of CoA plays a critical role in substrate recognition by PhaC_Re.

Keywords: Polyhydroxyalkanoate; PHA synthase; Kinetic analysis; Lag eliminator; TritonX-100

Adenosine kinase-deficient mutant of Saccharomyces cerevisiae accumulates S-adenosylmethionine because of an enhanced methionine biosynthesis pathway by Muneyoshi Kanai; Mitsunori Masuda; Yasumichi Takaoka; Hiroko Ikeda; Kazuo Masaki; Tsutomu Fujii; Haruyuki Iefuji (pp. 1183-1190).

To isolate an S-adenosylmethionine (SAM)-accumulating yeast strain and to develop a more efficient method of producing SAM, we screened methionine-resistant strains using the yeast deletion library of budding yeast and isolated 123 strains. The SAM content in 81 of the 123 strains was higher than that in the parental strain BY4742. We identified ADO1 encoding adenosine kinase as one of the factors participating in high SAM accumulation. The X∆ado1 strain that was constructed from the X2180-1A strain (MAT a, ATCC 26786) could accumulate approximately 30-fold (18 mg/g dry cell weight) more SAM than the X2180-1A strain in yeast extract peptone dextrose medium. Furthermore, we attempted to identify the molecular basis underlying the differences in SAM accumulation between X∆ado1 and X2180-1A strains. DNA microarray analysis revealed that the genes involved in the methionine biosynthesis pathway, phosphate metabolism, and hexose transport were mainly overexpressed in the X∆ado1 strain compared with the X2180-1A strain. We also determined the levels of various metabolites involved in the methionine biosynthesis pathway and found increased content of SAM, tetrahydrofolate (THF), inorganic phosphate, polyphosphoric acid, and S-adenosylhomocysteine in the X∆ado1 strain. In contrast, the content of 5-methyl-THF, homocysteine, glutathione, and adenosine was decreased. These results indicated that the ∆ado1 strain could accumulate SAM because of preferential activation of the methionine biosynthesis pathway.

Keywords: S-Adenosylmethionine; Adenosine kinase; Methionine biosynthesis pathway; Saccharomyces cerevisiae

Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation by Vijayalakshmi Kandasamy; Hema Vaidyanathan; Ivana Djurdjevic; Elamparithi Jayamani; K. B. Ramachandran; Wolfgang Buckel; Guhan Jayaraman; Subramanian Ramalingam (pp. 1191-1200).

Fermentation-derived products are in greater demand to meet the increasing global market as well as to overcome environmental problems. In this work, Escherichia coli has been metabolically engineered with acrylate pathway genes from Clostridium propionicum for the conversion of d-lactic acid to propionic acid. The introduced synthetic pathway consisted of seven genes encoding the enzymes propionate CoA-transferase (Pct), lactoyl-CoA dehydratase (Lcd) and acryloyl-CoA reductase (Acr). The engineered strain synthesised propionic acid at a concentration of 3.7 ± 0.2 mM upon fermentation on glucose. This low production level could be attributed to the low activity of the recombinant enzymes in particular the rate-limiting enzyme, Acr. Interestingly, the recombinant pathway caused an increased lactate production in E. coli with a yield of 1.9 mol/mol of glucose consumed along with a decrease in other by-products. Down-regulation of the pfl (pyruvate formate lyase) genes and a possible inhibition of Pfl activity by the acrylate pathway intermediate, acryloyl-CoA, could have reduced carbon flow to the Pfl pathway with a concomitant increase in lactate production. This study reports a novel way of synthesising propionic acid by employing a non-native, user-friendly organism through metabolic engineering.

Keywords: Acrylate pathway; Metabolic engineering; Pyruvate formate lyase; Lactic acid; E. coli

Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris by Carmen Menéndez; Duniesky Martínez; Luis E. Trujillo; Yuliet Mazola; Ernesto González; Enrique R. Pérez; Lázaro Hernández (pp. 1201-1212).

Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable β-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.

Keywords: Invertase; Fructosidase; Invert sugar; Pichia pastoris ; GAP promoter; Thermotoga maritima

Genome-based cryptic gene discovery and functional identification of NRPS siderophore peptide in Streptomyces peucetius by Hae-Min Park; Byung-Gee Kim; Dongsook Chang; Sailesh Malla; Hwang-Soo Joo; Eun-jung Kim; Sei-Jin Park; Jae Kyung Sohng; Pyoung Il Kim (pp. 1213-1222).

Identification of secondary metabolites produced by cryptic gene in bacteria may be difficult, but in the case of nonribosomal peptide (NRP)-type secondary metabolites, this study can be facilitated by bioinformatic analysis of the biosynthetic gene cluster and tandem mass spectrometry analysis. To illustrate this concept, we used mass spectrometry-guided bioinformatic analysis of genomic sequences to identify an NRP-type secondary metabolite from Streptomyces peucetius ATCC 27952. Five putative NRPS biosynthetic gene clusters were identified in the S. peucetius genome by DNA sequence analysis. Of these, the sp970 gene cluster encoded a complete NRPS domain structure, viz., C-A-T-C-A-T-E-C-A-T-C-A-T-C domains. Tandem mass spectrometry revealed that the functional siderophore peptide produced by this cluster had a molecular weight of 644.4 Da. Further analysis demonstrated that the siderophore peptide has a cyclic structure and an amino acid composition of AchfOrn–Arg–hOrn–hfOrn. The discovery of functional cryptic genes by analysis of the secretome, especially of NRP-type secondary metabolites, using mass spectrometry together with genome mining may contribute significantly to the development of pharmaceuticals such as hybrid antibiotics.

Keywords: Streptomyces peucetius ; Nonribosomal peptide synthetase; Genome mining; Siderophore peptide; Mass spectrometry

Expression, renaturation and biological activity of recombinant conotoxin GeXIVAWT by Bingmiao Gao; Dongting Zhangsun; Yong Wu; Bo Lin; Xiaopeng Zhu; Sulan Luo (pp. 1223-1230).

Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Conotoxin GeXIVAWT (CTX-GeXIVAWT) is a 28-amino acid peptide containing five cysteines isolated from the venom of Conus generalis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. The codes of novel conotoxin gene GeXIVAWT were optimized and generated two pairs of primers by chemical synthesis for construction of expression vector. Recombinant expression vector pET22b(+)-GeXIVAWT fused with pelB leader and His-tag was successfully expressed as an insoluble body in Escherichia coli BL21(DE3) cells. Recombinant conotoxin GeXIVAWT (rCTX-GeXIVAWT) was obtained by dissolving the insoluble bodies and purifying with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography and identified by matrix-assisted laser desorption/ionization–time of flight mass spectrometry. The rCTX-GeXIVAWT renatured in vitro could inhibited the growth of Sf9 cell with biological activity assay. This expression system may prove valuable for future structure–function studies of conotoxins.

Keywords: Conotoxin; Escherichia coli ; Recombinant expression; Purification

Protection against human papillomavirus type 16-induced tumors in mice using non-genetically modified lactic acid bacteria displaying E7 antigen at its surface by Pedro Ribelles; Bouasria Benbouziane; Philippe Langella; Juan E. Suárez; Luis G. Bermúdez-Humarán; Ali Riazi (pp. 1231-1239).

Human papillomavirus (HPV) is the causative agent of cervical cancer (CxCa) and the most commonly sexually transmitted pathogen worldwide. HPV type 16 (HPV-16) E7 oncoprotein is constitutively produced in CxCa and considered as a good antigen candidate for the development of new therapeutic CxCa vaccines. Here, we report the use of non-genetically modified, E7-expressing lactic acid bacteria (LAB) by using the cell-binding domain from Lactobacillus casei A2 phage lysin as a cell wall anchor. The versatility of this system was validated by investigating E7 stability at the surface of Lactococcus lactis and L. casei, two major species of LAB. Moreover, we demonstrated the successful use of these LAB displaying E7 antigen as a mucosal live vaccine in mice. Altogether, these results show the feasibility of using non-genetically modified LAB for low-cost mucosal immunotherapy against HPV-related CxCa in humans.

Keywords: Mucosal vaccines; Lactococcus lactis ; Lactobacillus casei ; Lactic acid bacteria; A2 phage lysin; Cell wall anchor; E7; HPV-16

Identification and deletion of the major secreted protein of Pichia pastoris by Silvia Heiss; Michael Maurer; Rainer Hahn; Diethard Mattanovich; Brigitte Gasser (pp. 1241-1249).

A major contaminating host cell protein was identified in fed batch cultures of Pichia pastoris producing an antibody Fab fragment. Purification and peptide sequencing identified this protein to be related to the cysteine-rich secretory protein family. The same protein was also observed as one of the most abundantly secreted proteins in chemostat cultures of a wild type P. pastoris strain. It has an apparent molecular weight of 65 kDa, 2-fold higher than predicted from the amino acid sequence, which is due to high O-glycosylation. It was denominated extracellular protein X 1 (Epx1), as no clear function could be attributed to it. The EPX1 gene is upregulated in different stress situations, and the respective deletion strain was more susceptible than the wild type to the cell wall damaging agents Calcofluor white and Congo red. The EPX1 deletion strain (Δepx1) was evaluated for its suitability for recombinant protein production. No significant difference in growth and product formation was observed between the wild type and the Δepx1 strain. Batch purification of a Fab fragment produced in the Δepx1 strain highlighted its superior purity due to the decreased host cell protein load.

Keywords: Pichia pastoris ; Extracellular protein; Host cell protein; Recombinant protein; Purification; CRISP family

Cloning, expression, and functional analysis of molecular motor pilT and pilU genes of type IV pili in Acidithiobacillus ferrooxidans by Yongquan Li; Shuangsheng Huang; Xiaosu Zhang; Tao Huang; Hongyu Li (pp. 1251-1257).

PilT is a hexameric ATPase required for type IV pili (Tfp) retraction in gram-negative bacterium. Retraction of Tfp mediates intimate attachment and motility on inorganic solid surfaces. We investigated the cloning and expression of pilT and pilU genes of Acidithiobacillus ferrooxidans strains ATCC 23270, and the results indicate that PilT and PilU contain the canonical conserved AIRNLIRE and GMQTXXXXLXXL motifs that are the characteristic motifs of the PilT protein family; PilT and PilU also contain the canonical nucleotide-binding motifs, named with Walker A box (GxxGxGKT/S) and Walker B box (hhhhDE), respectively. The pilT and pilU genes were expressed to produce 37.1- and 42.0-kDa proteins, respectively, and co-transcribed induced by 10 % mineral powder. However, ATPase activity of PilT was distinctly higher than those of PilU. These results indicated that the PilT protein was the real molecular motor of Tfp, while PilU could play a key role in the assembly, modification, and twitching motility of Tfp in A. ferrooxidans. However, PilT and PilU were nonetheless interrelated in the forming and function of the molecular motor of Tfp.

Keywords: Acidithiobacillus ferrooxidans ; Type IV pili; pilT ; pilU ; Functional analysis

Significance of the Cgl1427 gene encoding cytidylate kinase in microaerobic growth of Corynebacterium glutamicum by Seiki Takeno; Daisuke Shirakura; Nobuyoshi Tsukamoto; Satoshi Mitsuhashi; Masato Ikeda (pp. 1259-1267).

The Cgl1427 gene was previously found to be relevant to the microaerobic growth of Corynebacterium glutamicum (Ikeda et al. Biosci Biotechnol Biochem 73:2806–2808, 2009). In the present work, Cgl1427 was identified as a cytidylate kinase gene (cmk) by homology analysis of its deduced amino acid sequence with that of other bacterial cytidylate kinases (CMP kinases) and on the basis of findings that deletion of Cgl1427 results in loss of CMP kinase activity. Deletion of the cmk gene significantly impaired the growth of C. glutamicum in oxygen-limiting static culture, and the impaired growth was restored by introducing a plasmid containing the cmk gene, suggesting that this gene plays an important role in the microaerobic growth of C. glutamicum. On the other hand, in the main culture with aerobic shaking, a prolonged lag phase was observed in the cmk disruptant, despite an unchanged growth rate, compared to the behavior of the wild-type strain. The prolongation was observed when using seed culture grown to later growth stages in which oxygen limitation occurred, but it was not observed when using seed culture grown to an earlier growth stage in which oxygen remained relatively plentiful. Since nucleotide biosynthesis in C. glutamicum requires oxygen, we hypothesized that the ability of the cmk disruptant to synthesize nucleotides was influenced by oxygen limitation in the later growth stages of the seed culture, which caused the prolongation of the lag phase in the following shaken culture. To verify this hypothesis, a plasmid containing genes encoding all components of a homologous ribonucleotide reductase, a key enzyme for nucleotide synthesis that requires oxygen for its reaction, was introduced into the cmk disruptant, which significantly ameliorated the lag phase prolongation. Furthermore, this experimental setup almost completely restored the growth of the cmk disruptant in the oxygen-limiting static culture. These results indicate that CMP kinase plays an important role in normal nucleotide biosynthesis under an oxygen-limiting environment.

Keywords: Cytidylate kinase; Nucleotide synthesis; Oxygen; Microaerobic growth; Corynebacterium glutamicum

Monascus-fermented red mold rice exhibits cytotoxic effect and induces apoptosis on human breast cancer cells by Chu-I Lee; Chun-Lin Lee; Jyi-Faa Hwang; Yi-Hsin Lee; Jyh-Jye Wang (pp. 1269-1278).

Red mold rice (RMR) is a traditional food and folk medicine to Asian people and has recently become a popular health supplement. RMR has been shown to have some anticancer activities, although the mechanism for inducing cell death of human breast cancer cells is still not fully understood. In this study, bioactive extracts of RMR fermented by Monascus purpureus NTU 803 were analyzed for effects on apoptosis induction in human breast cancer cells. The RMR ethanol extract and ethyl acetate extract contain monacolin K, total phenols, and flavonoids, the three components that have been reported to have anticancer activity. Red mold rice extracts (RMRE) exhibited selective cytotoxic effect on MCF-7 cells. RMRE treatment induced apoptosis and cell cycle arrest at G2/M phase. Apoptosis was confirmed by annexin V–fluorescein isothiocyanate (FITC)/propidium iodide staining, the observation of characteristic chromatin condensation, nuclear DNA fragmentation, and poly(ADP-ribose) polymerase cleavage. Furthermore, the RMRE-induced apoptosis in MCF-7 cells may occur through a mitochondria-dependent pathway while triggering an appropriate balance of bax/bcl-2 and activation of caspase-9 and caspase-3 in a time-dependent manner. To conclude, RMRE exhibits direct cytotoxic and proapoptotic effects on MCF-7 cells and could be considered as a potential functional food for breast cancer prevention.

Keywords: Monascus purpureus ; Red mold rice; Monacolin K; Total phenols; Flavonoids; Apoptosis; Breast cancer cells

Induction of hexanol dehydrogenase in Geotrichum spp. by the addition of hexanol by Jinhua Zhang; Junling Shi; Hongliang Lv; Yanlin Liu (pp. 1279-1287).

Excessive hexanol content distorts the flavor of foods and is harmful to human health. Previously, two strains of fungi were found capable of producing hexanol-degrading enzymes. The current study identified these strains as Galactomyces geotrichum according to the gene sequence of the 26 S rDNA D1/D2 region (strain S12) and genus Geotrichum according to the gene sequence of ITS region (strain S13). Parallel analysis of extracellular and intracellular enzyme activities showed that the enzymes mainly accumulated intracellularly. Native polyacrylamide gel electrophoresis with reactive dyes showed the enzymes were alcohol dehydrogenases induced by the addition of hexanol. Hexanol was catalyzed into hexanoic acid and hexanal by strain S12 and into hexanoic acid by strain S13. The optimum conditions for the induction of enzymes were determined to be 6–9 h in the presence of 0.7 g/l hexanol. The identification of two strains capable of enzymatically degrading hexanol and optimum conditions for their induction will facilitate their use in industrial applications.

Keywords: 1-Hexanol; Alcohol dehydrogenase; Induction; Intracellular enzyme

Two different primary oxidation mechanisms during biotransformation of thymol by gram-positive bacteria of the genera Nocardia and Mycobacterium by Veronika Hahn; Katharina Sünwoldt; Annett Mikolasch; Frieder Schauer (pp. 1289-1297).

Thymol has antibacterial, antifungal, insecticidal, and antioxidative properties which are the basis for the wide use of this compound in the cosmetic, food, and pharmaceutical industries. Although thymol is a ubiquitously occurring substance in the environment, data about its degradation and detoxification by bacteria are sparse. Here, we show the existence of two different pathways for the biotransformation of thymol by Nocardia cyriacigeorgica and Mycobacterium neoaurum which were described for the first time for gram-positive bacteria. The first pathway starts with hydroxylation of thymol to thymohydroquinone (2-isopropyl-5-methylbenzene-1,4-diol) with subsequent oxidation to thymobenzoquinone (2-isopropyl-5-methyl-1,4-benzoquinone). The second pathway involves hydroxylation of the methyl group followed by oxidation to 3-hydroxy-4-isopropylbenzoic acid, possibly via the aldehyde 3-hydroxy-4-isopropylbenzaldehyde. It is noteworthy that the branched side chain of thymol was not oxidized. Similarities and differences of these oxidation processes with those of the gram-negative bacterium Pseudomonas putida, fungi, and plants are discussed and, in addition, the toxicity of thymol towards N. cyriacigeorgica and M. neoaurum was tested. The experiments showed a temporary growth inhibition with 0.025 % thymol. This was explained by degradation of thymol and the formation of products which are less toxic than thymol itself.

Keywords: Biotransformation; Degradation; Environmental pollution; Hydroxylation; Mineral oil; Quinone

Alteration of bacterial communities and organic matter in microbial fuel cells (MFCs) supplied with soil and organic fertilizer by Stefano Mocali; Carlo Galeffi; Elena Perrin; Alessandro Florio; Melania Migliore; Francesco Canganella; Giovanna Bianconi; Elena Di Mattia; Maria Teresa Dell’Abate; Renato Fani; Anna Benedetti (pp. 1299-1315).

The alteration of the organic matter (OM) and the composition of bacterial community in microbial fuel cells (MFCs) supplied with soil (S) and a composted organic fertilizer (A) was examined at the beginning and at the end of 3 weeks of incubation under current-producing as well as no-current-producing conditions. Denaturing gradient gel electrophoresis revealed a significant alteration of the microbial community structure in MFCs generating electricity as compared with no-current-producing MFCs. The genetic diversity of cultivable bacterial communities was assessed by random amplified polymorphic DNA (RAPD) analysis of 106 bacterial isolates obtained by using both generic and elective media. Sequencing of the 16S rRNA genes of the more representative RAPD groups indicated that over 50.4% of the isolates from MFCs fed with S were Proteobacteria, 25.1% Firmicutes, and 24.5% Actinobacteria, whereas in MFCs supplied with A 100% of the dominant species belonged to γ-Proteobacteria. The chemical analysis performed by fractioning the OM and using thermal analysis showed that the amount of total organic carbon contained in the soluble phase of the electrochemically active chambers significantly decreased as compared to the no-current-producing systems, whereas the OM of the solid phase became more humified and aromatic along with electricity generation, suggesting a significant stimulation of a humification process of the OM. These findings demonstrated that electroactive bacteria are commonly present in aerobic organic substrates such as soil or a fertilizer and that MFCs could represent a powerful tool for exploring the mineralization and humification processes of the soil OM.

Keywords: Microbial fuel cells; Soil; Organic matter; Electrogenic bacteria; Microbial diversity; Humification

Attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum cultured under varying conditions to pyrite, chalcopyrite, low-grade ore and quartz in a packed column reactor by Cindy-Jade Africa; Robert P. van Hille; Susan T. L. Harrison (pp. 1317-1324).

The attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum spp. grown on ferrous medium or adapted to a pyrite mineral concentrate to four mineral substrata, namely, chalcopyrite and pyrite concentrates, a low-grade chalcopyrite ore (0.5 wt%) and quartzite, was investigated. The quartzite represented a typical gangue mineral and served as a control. The attachment studies were carried out in a novel particle-coated column reactor. The saturated reactor containing glass beads, which were coated with fine mineral concentrates, provided a quantifiable surface area of mineral concentrate and maintained good fluid flow. A. ferrooxidans and Leptospirillum spp. had similar attachment characteristics. Enhanced attachment efficiency occurred with bacteria grown on sulphide minerals relative to those grown on ferrous sulphate in an ore-free environment. Selective attachment to sulphide minerals relative to gangue materials occurred, with mineral adapted cultures attaching to the minerals more efficiently than ferrous grown cultures. Mineral-adapted cultures showed highest levels of attachment to pyrite (74% and 79% attachment for A. ferrooxidans and L. ferriphilum, respectively). This was followed by attachment of mineral-adapted cultures to chalcopyrite (63% and 58% for A. ferrooxidans and L. ferriphilum, respectively). A. ferrooxidans and L. ferriphilum exhibited lower levels of attachment to low-grade ore and quartz relative to the sulphide minerals.

Keywords: Bioleaching; Attachment; Low-grade chalcopyrite; Pyrite; Acidithiobacillus ferrooxidans ; Leptospirillum ferriphilum

Minimizing nitrous oxide in biological nutrient removal from municipal wastewater by controlling copper ion concentrations by Xiaoyu Zhu; Yinguang Chen; Hong Chen; Xiang Li; Yongzhen Peng; Shuying Wang (pp. 1325-1334).

In this study, nitrous oxide (N₂O) production during biological nutrient removal (BNR) from municipal wastewater was reported to be remarkably reduced by controlling copper ion (Cu²⁺) concentration. Firstly, it was observed that the addition of Cu²⁺ (10–100 μg/L) reduced N₂O generation by 54.5–73.2 % and improved total nitrogen removal when synthetic wastewater was treated in an anaerobic–aerobic (with low dissolved oxygen) BNR process. Then, the roles of Cu²⁺ were investigated. The activities of nitrite and nitrous oxide reductases were increased by Cu²⁺ addition, which accelerated the bio-reductions of both nitrite to nitric oxide (NO ₂ ⁻ → NO) and nitrous oxide to nitrogen gas (N₂O → N₂). The quantitative real-time polymerase chain reaction assay indicated that Cu²⁺ addition increased the number of N₂O reducing denitrifiers. Further investigation showed that more polyhydoxyalkanoates were utilized in the Cu²⁺-added system for denitrification. Finally, the feasibility of reducing N₂O generation by controlling Cu²⁺ was examined in two other BNR processes treating real municipal wastewater. As the Cu²⁺ in municipal wastewater is usually below 10 μg/L, according to this study, the supplement of influent Cu²⁺ to a concentration of 10–100 μg/L is beneficial to reduce N₂O emission and improve nitrogen removal when sludge concentration in the BNR system is around 3,200 mg/L.

Keywords: Nitrous oxide; Reduction; Cu²⁺ ; Wastewater; Biological nutrient removal

Influence of temperature on the effectiveness of a biogenic carbonate surface treatment for limestone conservation by Willem De Muynck; Kim Verbeken; Nele De Belie; Willy Verstraete (pp. 1335-1347).

So far, most studies on microbiologically induced carbonate precipitation for limestone conservation have been performed at temperatures optimal for the activity of the calcinogenic bacteria (i.e., 20–28 °C). Successful application in practice, however, requires adequate performance in a wide range of environmental conditions. Therefore, the aim of this study was to select microorganisms that are most suited for biodeposition at temperatures relevant for practice. In a first step, ureolytic microorganisms were screened for their growth and ureolytic activity at different temperatures (10, 20, 28, and 37 °C). Large differences in calcinogenic activity could be observed between experiments performed on agar plates and those performed in solution and in limestone. In a second step, the influence of temperature on the performance of the biodeposition treatment with different ureolytic microorganisms was evaluated, both on the consolidative and protective effect of the treatment. In contrast with the experiments on agar plates, the Sporosarcina psychrophila strains failed to produce significant amounts of calcium carbonate on limestone in conditions relevant for practice, even at 10 °C. This resulted in a poor performance of the treatment. From experiments performed on limestone prisms, it appeared that the mesophilic Bacillus sphaericus produced the highest amount of carbonate in the shortest amount of time at all temperatures tested. As a result, compared to the untreated specimens, the highest consolidative (64 % lower weight loss upon sonication) and protective effect (46 % decreased sorptivity) were observed for the treatments with this species. From this study, it appears that among all ureolytic strains tested, B. sphaericus is most suited for biodeposition applications in practice.

Keywords: Sporosarcina ; Bacillus ; Biomineralization; Urea; Bacteria; Stone

Denitrification in soil amended with thermophile-fermented compost suppresses nitrate accumulation in plants by Kazuto Ishikawa; Takashi Ohmori; Hirokuni Miyamoto; Toshiyuki Ito; Yoshifumi Kumagai; Masatoshi Sonoda; Jirou Matsumoto; Hisashi Miyamoto; Hiroaki Kodama (pp. 1349-1359).

NO ₃ ⁻ is a major nitrogen source for plant nutrition, and plant cells store NO ₃ ⁻ in their vacuoles. Here, we report that a unique compost made from marine animal resources by thermophiles represses NO ₃ ⁻ accumulation in plants. A decrease in the leaf NO ₃ ⁻ content occurred in parallel with a decrease in the soil NO ₃ ⁻ level, and the degree of the soil NO ₃ ⁻ decrease was proportional to the compost concentration in the soil. The compost-induced reduction of the soil NO ₃ ⁻ level was blocked by incubation with chloramphenicol, indicating that the soil NO ₃ ⁻ was reduced by chloramphenicol-sensitive microbes. The compost-induced denitrification activity was assessed by the acetylene block method. To eliminate denitrification by the soil bacterial habitants, soil was sterilized with γ irradiation and then compost was amended. After the 24-h incubation, the N₂O level in the compost soil with presence of acetylene was approximately fourfold higher than that in the compost soil with absence of acetylene. These results indicate that the low NO ₃ ⁻ levels that are often found in the leaves of organic vegetables can be explained by compost-mediated denitrification in the soil.

Keywords: Compost; Denitrification; Organic food; Plant nitrate concentration; Thermophile

Rhizocompetence and antagonistic activity towards genetically diverse Ralstonia solanacearum strains – an improved strategy for selecting biocontrol agents by Qing-Yun Xue; Guo-Chun Ding; Shi-Mo Li; Yang Yang; Cheng-Zhong Lan; Jian-Hua Guo; Kornelia Smalla (pp. 1361-1371).

Bacterial wilt caused by Ralstonia solanacearum is a serious threat for agricultural production in China. Eight soil bacterial isolates with activity against R. solanacearum TM15 (biovar 3) were tested in this study for their in vitro activity towards ten genetically diverse R. solanacearum isolates from China. The results indicated that each antagonist showed remarkable differences in its ability to in vitro antagonize the ten different R. solanacearum strains. Strain XY21 (based on 16S rRNA gene sequencing affiliated to Serratia) was selected for further studies based on its in vitro antagonistic activity and its excellent rhizocompetence on tomato plants. Under greenhouse conditions XY21 mediated biocontrol of tomato wilt caused by seven different R. solanacearum strains ranged from 19 to 70 %. The establishment of XY21 and its effects on the bacterial community in the tomato rhizosphere were monitored by denaturing gradient gel electrophoresis of 16S rRNA gene fragments PCR-amplified from total community DNA. A positive correlation of the in vitro antagonistic activities of XY21 and the actual biocontrol efficacies towards seven genetically different R. solanacearum strains was found and further confirmed by the efficacy of XY21 in controlling bacterial wilt under field conditions.

Keywords: Ralstonia solanacearum ; Biocontrol; Serratia sp.; Rhizocompetence; Pathogen diversity; DGGE

Co-digestion of manure and whey for in situ biogas upgrading by the addition of H₂: process performance and microbial insights by Gang Luo; Irini Angelidaki (pp. 1373-1381).

In situ biogas upgrading was conducted by introducing H₂ directly to the anaerobic reactor. As H₂ addition is associated with consumption of the CO₂ in the biogas reactor, pH increased to higher than 8.0 when manure alone was used as substrate. By co-digestion of manure with acidic whey, the pH in the anaerobic reactor with the addition of hydrogen could be maintained below 8.0, which did not have inhibition to the anaerobic process. The H₂ distribution systems (diffusers with different pore sizes) and liquid mixing intensities were demonstrated to affect the gas-liquid mass transfer of H₂ and the biogas composition. The best biogas composition (75:6.6:18.4) was obtained at stirring speed 150 rpm and using ceramic diffuser, while the biogas in the control reactor consisted of CH₄ and CO₂ at a ratio of 55:45. The consumed hydrogen was almost completely converted to CH₄, and there was no significant accumulation of VFA in the effluent. The study showed that addition of hydrogen had positive effect on the methanogenesis, but had no obvious effect on the acetogenesis. Both hydrogenotrophic methanogenic activity and the concentration of coenzyme F₄₂₀ involved in methanogenesis were increased. The archaeal community was also altered with the addition of hydrogen, and a Methanothermobacter thermautotrophicus related band appeared in a denaturing gradient gel electrophoresis gel from the sample of the reactor with hydrogen addition. Though the addition of hydrogen increased the dissolved hydrogen concentration, the degradation of propionate was still thermodynamically feasible at the reactor conditions.

Keywords: Anaerobic digestion; Co-digestion; Hydrogen; In situ biogas upgrading

Carotenoid and lipid production by the autotrophic microalga Chlorella protothecoides under nutritional, salinity, and luminosity stress conditions by L. Campenni’; B. P. Nobre; C. A. Santos; A. C. Oliveira; M. R. Aires-Barros; A. M. F. Palavra; L. Gouveia (pp. 1383-1393).

Today microalgae represent a viable alternative source for high-value products. The specie Chlorella protothecoides (Cp), heterotrophically grown, has been widely studied and provides a high amount of lutein and fatty acids (FA) and has a good profile for biodiesel production. This work studies carotenoid and FA production by autotrophic grown Cp. Cp was grown until the medium’s nitrogen was depleted, then diluted in NaCl solution, resulting in nutritional, luminosity, and salinity stresses. Different NaCl concentrations were tested (10, 20, 30 g/L) at two different dilutions. After dilution, a color shifting from green to orange-red was noticed, showing carotenoid production. The best production of both carotenoids and FA was attained with a 20 g/L NaCl solution. The total carotenoid content was 0.8 % w/w (canthaxanthin (23.3 %), echinenone (14.7 %), free astaxanthin (7.1 %), and lutein/zeaxanthin (4.1 %)). Furthermore, the total lipid content reached 43.4 % w/w, with a FA composition of C18:1 (33.64 %), C16:0 (23.30 %), C18:2 (11.53 %), and less than 12 % of C18:3, which is needed to fulfill the biodiesel quality specifications (EN 14214).

Keywords: Chlorella protothecoides ; Microalga; Autotrophic; Carotenoids; Fatty acids; Biodiesel

Erratum to: Protection against human papillomavirus type 16-induced tumors in mice using non-genetically modified lactic acid bacteria displaying E7 antigen at its surface by Pedro Ribelles; Bouasria Benbouziane; Philippe Langella; Juan E. Suárez; Luis G. Bermúdez-Humarán (pp. 1395-1395).

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Applied Microbiology and Biotechnology (v.97, #3)

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Applied Microbiology and Biotechnology (v.97, #3)