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Applied Microbiology and Biotechnology (v.97, #4)

Protein-based biorefining: metabolic engineering for production of chemicals and fuel with regeneration of nitrogen fertilizers by David G. Wernick; James C. Liao (pp. 1397-1406).

Threats to stable oil supplies and concerns over environmental emissions have pushed for renewable biofuel developments to minimize dependence on fossil resources. Recent biofuel progress has moved towards fossil resource-independent carbon cycles, but environmental issues regarding use of nitrogen fertilizers have not been addressed on a global scale. The recently demonstrated conversion of waste protein biomass into advanced biofuels and renewable chemicals, while recycling nitrogen fertilizers, offers a glimpse of the efforts needed to balance the nitrogen cycle at scale. In general, the catabolism of protein into biofuels is challenging because of physiological regulation and thermodynamic limitations. This conversion became possible with metabolic engineering around ammonia assimilation, intracellular nitrogen flux, and quorum sensing. This review highlights the metabolic engineering solutions in transforming those cellular processes into driving forces for the high yield of chemical products from protein.

Keywords: Biofuels; Metabolic engineering; Nitrogen fertilizer; Ammonia recycling; Protein conversion

Microbial production of poly(hydroxybutyrate) from C₁ carbon sources by Kianoush Khosravi-Darani; Zahra-Beigom Mokhtari; Tomohito Amai; Kenji Tanaka (pp. 1407-1424).

Polyhydroxybutyrate (PHB) is an attractive substitute for petrochemical plastic due to its similar properties, biocompatibility, and biodegradability. The cost of scaled-up PHB production inhibits its widespread usage. Intensive researches are growing to reduce costs and improve thermomechanical, physical, and processing properties of this green biopolymer. Among cheap substrates which are used for reducing total cost of PHB production, some C₁ carbon sources, e.g., methane, methanol, and CO₂ have received a great deal of attention due to their serious role in greenhouse problem. This article reviews the fundamentals of strategies for reducing PHA production and moves on to the applications of several cheap substrates with a special emphasis on methane, methanol, and CO₂. Also, some explanation for involved microorganisms including the hydrogen-oxidizing bacteria and methanotrophs, their history, culture condition, and nutritional requirements are given. After description of some important strains among the hydrogen-oxidizing and methanotrophic producers of PHB, the article is focused on limitations, threats, and opportunities for application and their future trends.

Keywords: Polyhydroxybutyrate; C₁ carbon sources; Methanol; Methane; CO₂

Microbial production of poly(hydroxybutyrate) from C₁ carbon sources by Kianoush Khosravi-Darani; Zahra-Beigom Mokhtari; Tomohito Amai; Kenji Tanaka (pp. 1407-1424).

Keywords: Polyhydroxybutyrate; C₁ carbon sources; Methanol; Methane; CO₂

amoA-encoding archaea in wastewater treatment plants: a review by Tawan Limpiyakorn; Maria Fürhacker; Raimund Haberl; Thanasita Chodanon; Papitchaya Srithep; Puntipar Sonthiphand (pp. 1425-1439).

Recent evidence from natural environments suggests that in addition to ammonia-oxidizing bacteria, ammonia-oxidizing archaea (AOA) affiliated with Thaumarcheota, a new phylum of the domain Archaea, also oxidize ammonia to nitrite and thus participate in the global nitrogen cycle. Besides natural environments, modern data indicate the presence of amoA-encoding archaea (AEA) in wastewater treatment plants (WWTPs). To further elucidate whether AEA in WWTPs are AOA and to clarify the role of AEA in WWTPs, this paper reviews the current knowledge on this matter for wastewater engineers and people in related fields. The initial section coveys a microbiological point of view and is particularly based upon data from AOA cultures. The later section summarizes what is currently known about AEA in relation to WWTPs. Based on the reviewed data, future research pathways are proposed in an effort to further what is known about AEA in wastewater treatment systems.

Keywords: Ammonia-oxidizing archaea; Ammonia-oxidizing bacteria; amoA-encoding archaea; Thaumarcheota; Wastewater treatment plant

Biocatalysis with immobilized Escherichia coli by Petra Zajkoska; Martin Rebroš; Michal Rosenberg (pp. 1441-1455).

Immobilization is one of the great tools for developing economically and ecologically available biocatalysts and can be applied for both enzymes and whole cells. Much research dealing with the immobilization of Escherichia coli has been published in the past two decades. E. coli in the form of immobilized biocatalyst catalyzes many interesting reactions and has been used mainly in laboratories, but also on an industrial scale, leading to the production of valuable substances. It has the potential to be applied in many fields of modern biotechnology. This paper aims to give a general overview of immobilization techniques and matrices suitable mostly for entrapment, encapsulation, and adsorption, which have been most frequently used for the immobilization of E. coli. An extensive analysis reviewing the history and current state of immobilized E. coli catalyzing different types of biotransformations is provided. The review is organized according to the enzymes expressed in immobilized E. coli, which were grouped into main enzyme classes. The industrial applications of immobilized E. coli biocatalyst are also discussed.

Keywords: Immobilization; E. coli ; Biocatalysis; Biotransformation

Biotechnological production of ethanol from renewable resources by Neurospora crassa: an alternative to conventional yeast fermentations? by Ioannis Dogaris; Diomi Mamma; Dimitris Kekos (pp. 1457-1473).

Microbial production of ethanol might be a potential route to replace oil and chemical feedstocks. Bioethanol is by far the most common biofuel in use worldwide. Lignocellulosic biomass is the most promising renewable resource for fuel bioethanol production. Bioconversion of lignocellulosics to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/distillation. Conventional bioethanol processes for lignocellulosics apply commercial fungal cellulase enzymes for biomass hydrolysis, followed by yeast fermentation of resulting glucose to ethanol. The fungus Neurospora crassa has been used extensively for genetic, biochemical, and molecular studies as a model organism. However, the strain's potential in biotechnological applications has not been widely investigated and discussed. The fungus N. crassa has the ability to synthesize and secrete all three enzyme types involved in cellulose hydrolysis as well as various enzymes for hemicellulose degradation. In addition, N. crassa has been reported to convert to ethanol hexose and pentose sugars, cellulose polymers, and agro-industrial residues. The combination of these characteristics makes N. crassa a promising alternative candidate for biotechnological production of ethanol from renewable resources. This review consists of an overview of the ethanol process from lignocellulosic biomass, followed by cellulases and hemicellulases production, ethanol fermentations of sugars and lignocellulosics, and industrial application potential of N. crassa.

Keywords: Neurospora crassa ; Ethanol; Fermentation; Lignocellulosic; Cellulases; Hemicellulases

Recent discoveries and applications of Anoxybacillus by Kian Mau Goh; Ummirul Mukminin Kahar; Yen Yen Chai; Chun Shiong Chong; Kian Piaw Chai; Velayudhan Ranjani; Rosli Md. Illias; Kok-Gan Chan (pp. 1475-1488).

The Bacillaceae family members are a good source of bacteria for bioprocessing and biotransformation involving whole cells or enzymes. In contrast to Bacillus and Geobacillus, Anoxybacillus is a relatively new genus that was proposed in the year 2000. Because these bacteria are alkali-tolerant thermophiles, they are suitable for many industrial applications. More than a decade after the first report of Anoxybacillus, knowledge accumulated from fundamental and applied studies suggests that this genus can serve as a good alternative in many applications related to starch and lignocellulosic biomasses, environmental waste treatment, enzyme technology, and possibly bioenergy production. This current review provides the first summary of past and recent discoveries regarding the isolation of Anoxybacillus, its medium requirements, its proteins that have been characterized and cloned, bioremediation applications, metabolic studies, and genomic analysis. Comparisons to some other members of Bacillaceae and possible future applications of Anoxybacillus are also discussed.

Keywords: Bacillaceae ; Bacillus ; Biomass; Bioremediation; Geobacillus ; Thermophile

Beneficial effects of phytoestrogens and their metabolites produced by intestinal microflora on bone health by Shen-Shih Chiang; Tzu-Ming Pan (pp. 1489-1500).

Phytoestrogens are a class of bioactive compounds derived from plants and exert various estrogenic and antiestrogenic effects. Estrogen deficiency osteoporosis has become a serious problem in elderly women. The use of ovariectomized (OVX) rat or mice models to simulate the postmenopausal condition is well established. This review aimed to clarify the sources, biochemistry, absorption, metabolism, and mode of action of phytoestrogens on bone health in intervention studies. In vitro, phytoestrogens promote protein synthesis, osteoprotegerin/receptor activation of nuclear factor-kappa B ligand ratio, and mineralization by osteoblast-like cells (MC3T3-E1). In the OVX murine model, administration of phytoestrogens can inhibit differentiation and activation of osteoclasts, expression of tartrate-resistant acid phosphatase, and secretion of pyridinoline compound. Phytoestrogens also enhance bone formation and increase bone mineral density and levels of alkaline phosphatase, osteocalcin, osteopontin, and α1(I) collagen. Results of mechanistic studies have indicated that phytoestrogens suppress the rate of bone resorption and enhance the rate of bone formation.

Keywords: Phytoestrogens; Postmenopausal; Ovariectomized model; Antiosteoporosis

Bioconversion of p-coumaric acid to p-hydroxystyrene using phenolic acid decarboxylase from B. amyloliquefaciens in biphasic reaction system by Da-Hye Jung; Wonji Choi; Kwon-Young Choi; Eunok Jung; Hyungdon Yun; Romas J. Kazlauskas; Byung-Gee Kim (pp. 1501-1511).

Phenolic acid decarboxylase (PAD) catalyzes the non-oxidative decarboxylation of p-coumaric acid (pCA) to p-hydroxystyrene (pHS). PAD from Bacillus amyloliquefaciens (BAPAD), which showed k _cat/K _m value for pCA (9.3 × 10³ mM⁻¹ s⁻¹), was found as the most active one using the “Subgrouping Automata” program and by comparing enzyme activity. However, the production of pHS of recombinant Escherichia coli harboring BAPAD showed only a 22.7 % conversion yield due to product inhibition. Based on the partition coefficient of pHS and biocompatibility of the cell, 1-octanol was selected for the biphasic reaction. The conversion yield increased up to 98.0 % and 0.83 g/h/g DCW productivity was achieved at 100 mM pCA using equal volume of 1-octanol as an organic solvent. In the optimized biphasic reactor, using a three volume ratio of 1-octanol to phosphate buffer phase (50 mM, pH 7.0), the recombinant E. coli produced pHS with a 88.7 % conversion yield and 1.34 g/h/g DCW productivity at 300 mM pCA.

Keywords: Phenolic acid decarboxylase; Bioconversion; p-Coumaric acid; p-Hydroxystyrene; Biphasic reaction; 1-Octanol

Polyaniline-assisted silver nanoparticles: a novel support for the immobilization of α-amylase by Mohammad Jahir Khan; Qayyum Husain; Sajid Ali Ansari (pp. 1513-1522).

The size distribution of nanomaterials and their similarity in size with enzyme molecules together with other advantageous properties such as thermal stability, high surface-to-volume ratio, and irradiation resistance have revolutionized nanobiocatalytic approaches in various areas of enzyme technology. In the present study, polyaniline-assisted Ag nanocomposites were synthesized using ammonium peroxydisulfate as oxidant. These nanocomposites were used as a support for the covalent conjugation of α-amylase, one of the important industrial enzymes. X-ray diffraction study showed that the crystalline nature of nanocomposites was increased in the presence of Ag nanoparticles. Thermogravimetric and differential thermal analysis revealed that the synthesized nanocomposites retained significantly very high thermal stability. Scanning electron micrograph showed that Ag nanoparticles were homogeneously dispersed in polyaniline film providing large surface area and microenvironment for enzyme loading. Fourier transform infrared spectroscopy confirmed the conjugation of α-amylase to the functionalized nanocomposites. The conjugated α-amylase exhibited better tolerance to variations in the medium pH and temperature compared with the native enzyme. Immobilized α-amylase hydrolyzes starch more efficiently as compared to the free enzyme in batch process.

Keywords: α-Amylase; Immobilization; Polyaniline; Nanocomposites; Starch hydrolysis

Photosynthetic efficiency and oxygen evolution of Chlamydomonas reinhardtii under continuous and flashing light by Carsten Vejrazka; Marcel Janssen; Giulia Benvenuti; Mathieu Streefland; René H. Wijffels (pp. 1523-1532).

As a result of mixing and light attenuation in a photobioreactor (PBR), microalgae experience light/dark (L/D) cycles that can enhance PBR efficiency. One parameter which characterizes L/D cycles is the duty cycle; it determines the time fraction algae spend in the light. The objective of this study was to determine the influence of different duty cycles on oxygen yield on absorbed light energy and photosynthetic oxygen evolution. Net oxygen evolution of Chlamydomonas reinhardtii was measured for four duty cycles (0.05, 0.1, 0.2, and 0.5) in a biological oxygen monitor (BOM). Oversaturating light flashes were applied in a square-wave fashion with four flash frequencies (5, 10, 50, and 100 Hz). Algae were precultivated in a turbidostat and acclimated to a low photon flux density (PFD). A photosynthesis–irradiance (PI) curve was measured under continuous illumination and used to calculate the net oxygen yield, which was maximal between a PFD of 100 and 200 μmol m⁻² s⁻¹. Net oxygen yield under flashing light was duty cycle-dependent: the highest yield was observed at a duty cycle of 0.1 (i.e., time-averaged PFD of 115 μmol m⁻² s⁻¹). At lower duty cycles, maintenance respiration reduced net oxygen yield. At higher duty cycles, photon absorption rate exceeded the maximal photon utilization rate, and, as a result, surplus light energy was dissipated which led to a reduction in net oxygen yield. This behavior was identical with the observation under continuous light. Based on these data, the optimal balance between oxygen yield and production rate can be determined to maximize PBR productivity.

Keywords: Microalgae; Chlamydomonas reinhardtii ; Photosynthetic efficiency; Quantum yield; Flashing light; PI curve

Critical factors for the replication of mumps virus in primary chicken embryo fibroblasts defined by the use of design of experiments (DoE) by Maja Markusic; Nediljko Pavlović; Maja Šantak; Gorana Marić; Ljerka Kotarski; Dubravko Forcic (pp. 1533-1541).

Live attenuated vaccines against mumps virus (MuV) have been traditionally produced by passaging the virus in the embryonated chicken eggs or primary chicken embryo fibroblasts (CEFs). Virus propagation on these cell substrates enables successful virus attenuation and retains it sufficiently antigenic to induce lasting protective immunity in humans. The aim of this study was to identify critical factors for MuV replication in primary CEFs grown on a small-scale level in order to explore possibilities for improvements in the virus replication and yield. The effect of differently prepared cells, culturing conditions, and infection conditions on virus yield was estimated by employing statistical design of experiments (DoE) methodology. Our results show that the preparation of primary CEFs and the way of their infection substantially impact virus yield and are critical for efficient MuV replication. These process parameters should be considered in further process optimization. We also demonstrate the applicability of DoE in optimization of virus replication as a crucial step in obtaining high virus yields.

Keywords: Mumps virus; Primary chicken embryo fibroblasts; Design of experiments; Replication; Yield

Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro by Karine Rech Begnini; Caroline Rizzi; Vinicius Farias Campos; Sibele Borsuk; Eduarda Schultze; Virginia Campello Yurgel; Fernanda Nedel; Odir Antônio Dellagostin; Tiago Collares; Fabiana Kömmling Seixas (pp. 1543-1552).

BCG therapy remains at the forefront of immunotherapy for treating patients with superficial bladder cancer. The high incidence of local side effects and the presence of non-responder diseases have led to efforts to improve the therapy. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ∆leuD/Ag85B), could enhance the cytotoxicity to the human bladder carcinoma cell line 5637. The rBCG was generated using an expression plasmid encoding the mycobacterial antigen Ag85B to transform a BCG ∆leuD strain. The inhibitory effect of BCG ∆leuD/Ag85B on 5637 cells was determined by the MTT method, morphology observation and a LIVE/DEAD assay. Gene expression profiles for apoptotic, cell cycle-related and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ∆leuD/Ag85B treatment was evaluated by Western blotting. BCG ∆leuD/Ag85B revealed a superior cytotoxicity effect compared to the control strains used in this study. The results showed that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ∆leuD/Ag85B treatment, whereas the mRNA levels of anti-apoptotic genes decreased. Interestingly, BCG ∆leuD/Ag85B also increased the mRNA level of antioxidant enzymes in the bladder cancer cell line. Bax and p53 proteins levels increased following treatment. In conclusion, these results suggest that treatment with BCG ∆leuD/Ag85B enhances cytotoxicity for superficial bladder cancer cells in vitro. Therefore, rBCG therapy may have potential benefits in the treatment of bladder cancer.

Keywords: Bacillus Calmette-Guérin; Recombinant BCG; Superficial bladder cancer; Antitumor activity

Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody by Yanping Tian; Wei Chen; Ya Yang; Xiaofeng Xu; Junlei Zhang; Jiali Wang; Lan Xiao; Zongtao Chen (pp. 1553-1560).

Dengue virus is a major international public health concern, and there is a lack of available effective vaccines. Virus-specific epitopes could help in developing epitope peptide vaccine. Previously, a neutralizing monoclonal antibody (mAb) 4F5 against nonstructural protein 3 (NS3) of dengue virus 2 (DV2) was developed in our lab. In this work, the B cell epitope recognized by mAb 4F5 was identified using the phage-displayed peptide library. The results of the binding assay and competitive inhibition assay indicated that the peptides, residues 460–469 (U460-469 RVGRNPKNEN) of DV2 NS3 protein, were the B cell epitopes recognized by mAb 4F5. Furthermore, the epitope peptides and a control peptide were synthesized and then immunized female BALB/c mice. ELISA analysis showed that immunization with synthesized epitope peptide elicited a high level of antibody in mice, and immunofluorescent staining showed that the antisera from fusion epitope-immunized mice also responded to DV2 NS3 protein, which further characterized the specific response of the present epitope peptide. Therefore, the present work revealed the specificity of the newly identified epitope (U460-469) of DV2 NS3 protein, which may shed light on dengue virus (DV) vaccine design, DV pathogenesis study, and even DV diagnostic reagent development.

Keywords: Dengue virus; NS3 protein; Epitope; Vaccine

Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli by Won-Heong Lee; Jin-Woo Kim; Eun-Hee Park; Nam Soo Han; Myoung-Dong Kim; Jin-Ho Seo (pp. 1561-1569).

Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-l-fucose and ε-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E. coli producing GDP-l-fucose resulted in a maximum GDP-l-fucose concentration of 291.5 mg/l, which corresponded to a 51 % enhancement compared with the control strain. In a fed-batch Baeyer–Villiger (BV) oxidation of cyclohexanone using recombinant E. coli expressing Pos5p, a maximum ε-caprolactone concentration of 21.6 g/l was obtained, which corresponded to a 96 % enhancement compared with the control strain. Such an increase might be due to the enhanced availability of NADPH in recombinant E. coli expressing Pos5p. These results suggested that efficient regeneration of NADPH was possible by functional expression of Pos5p in recombinant E. coli, which can be applied to other NADPH-dependent biotransformation processes in E. coli.

Keywords: Recombinant Escherichia coli ; Cofactor; NADH kinase; GDP-l-fucose; ε-caprolactone; Fed-batch fermentation

Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange by Gabriele Siedenburg; Michael Breuer; Dieter Jendrossek (pp. 1571-1580).

Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC Aac), Acetobacter pasteurianus (SHC Apa), Streptomyces coelicolor (SHC Sco) and Bradyrhizobium japonicum (SHC Bja) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.

Keywords: Squalene-hopene cyclases; Monoterpenes; Citronellal; Menthol

An efficient lipase-catalyzed enantioselective hydrolysis of (R,S)-azolides derived from N-protected proline, pipecolic acid, and nipecotic acid by Chia-Hui Wu; Chia-Wen Pen; Pei-Yun Wang; Shau-Wei Tsai (pp. 1581-1587).

In the Candida antarctica lipase B-catalyzed hydrolysis of (R,S)-azolides derived from (R,S)-N-protected proline in water-saturated methyl tert-butyl ether (MTBE), high enzyme activity with excellent enantioselectivity (V _S V _R ⁻¹ > 100) for (R,S)-N-Cbz-proline 1,2,4-triazolide (1) and (R,S)-N-Cbz-proline 4-bromopyrazolide (2) was exploited in comparison with their corresponding methyl ester analog (3). Changing of the substrate structure, water content, solvent, and temperature was found to have profound influences on the lipase performance. On the basis of enzyme activity and enantioselectivity and solvent boiling point, the best reaction condition of using 1 as the substrate in water-saturated MTBE at 45 °C was selected and further employed for the successful resolution of (R,S)-N-Cbz-pipecolic 1,2,4-triazolide (5) and (R,S)-N-Boc-nipecotic 1,2,4-triazolide (9). Moreover, more than 89.1 % recovery of remained (R)-1 is obtainable in five cycles of enzyme reusage, when pH 7 phosphate buffers were employed as the extract at 4 °C.

Keywords: Lipase; Hydrolytic resolution; Azolides; N-Protected heterocyclic amino acids

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus by Kristiina Hildén; Miia R. Mäkelä; Taina Lundell; Jaana Kuuskeri; Alexey Chernykh; Ludmila Golovleva; David B. Archer; Annele Hatakka (pp. 1589-1599).

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.

Keywords: Laccase; Phenol oxidase; Thermotolerance; White-rot fungi; Physisporinus rivulosus ; Basidiomycete; 3-D homology modelling; Wood decay; Lignin degradation

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential by Lakshmi Prasanna; Vincent G. H. Eijsink; Richard Meadow; Sigrid Gåseidnes (pp. 1601-1611).

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.

Keywords: Brevibacillus laterosporus ; chitinase; Diamondback moth; Plutella xylostella

Molecular cloning, characterization, and engineering of xylitol dehydrogenase from Debaryomyces hansenii by Dipanwita Biswas; Manish Datt; Monika Aggarwal; Alok K. Mondal (pp. 1613-1623).

Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.

Keywords: Xylitol dehydrogenase; Medium-chain dehydrogenase reductase; Debaryomyces hansenii ; Halotolerant yeast; Protein engineering

Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s by Marco Girhard; Florian Tieves; Evelyne Weber; Martha Sophia Smit; Vlada B. Urlacher (pp. 1625-1635).

Candida apicola belongs to a group of yeasts producing surface-active glycolipids consisting of sophorose and long-chain (ω)- or (ω-1)-hydroxy fatty acids. Hydroxylation of the fatty acids in this strain is likely catalyzed by cytochrome P450 monooxygenases (P450), which require reducing equivalents delivered via a cytochrome P450-diflavin reductase (CPR). We herein report cloning and characterization of the cpr gene from C. apicola ATCC 96134. The gene encoding a protein of 687 amino acids was cloned in Escherichia coli and the enzyme was expressed in functional form after truncation of its N-terminal putative membrane anchor. The truncated recombinant protein showed cytochrome c reducing activity (K _M of 13.8 μM and k _cat of 1,915 per minute). Furthermore, we herein demonstrate to our best knowledge for the first time the use of a eukaryotic CPR to transfer electrons to bacterial P450s (namely CYP109B1 and CYP154E1). Cloning and characterization of this CPR therefore is not only an important step in the study of the P450 systems of C. apicola, but also provides a versatile redox partner for the characterization of other bacterial P450s with appealing biotechnological potential. The GenBank accession number of the sequence described in this article is JQ015264.

Keywords: Candida apicola ; Cytochrome P450-diflavin reductase; CPR; P450; Heterologous expression

Characterization and immobilization of a novel SGNH hydrolase (Est24) from Sinorhizobium meliloti by Song Yi Bae; Bum Han Ryu; Eunjin Jang; Seulgi Kim; T. Doohun Kim (pp. 1637-1647).

A novel oligomeric SGNH hydrolase (Est24) from Sinorhizobium meliloti was identified, actively expressed in Escherichia coli, characterized, and immobilized for industrial application. Sequence analysis of Est24 revealed a putative catalytic triad (Ser¹³-Asp¹⁶³-His¹⁶⁹), with moderate homology to other SGNH hydrolases. Est24 was more active toward short-chain esters, such as p-nitrophenyl acetate, butyrate, and valerate, while the S13A mutant completely lost its activity. Moreover, the activity of Est24 toward α- and β-naphthyl acetate, and enantioselectivity on (R)- and (S)-methyl-3-hydroxy-2-methylpropionate were tested. Est24 exhibited optimum activity at mesophilic temperature ranges (45–55 °C), and slightly alkaline pH (8.0). Structural and mutagenesis studies revealed critical residues involved in the formation of a catalytic triad and substrate-binding pocket. Cross-linked enzyme aggregates (CLEAs) of Est24 with and without amyloid fibrils were prepared, and amyloid fibril-linked Est24 with amyloid fibrils retained 83 % of its initial activity after 1 h of incubation at 60 °C. The high thermal stability of immobilized Est24 highlights its potential in the pharmaceutical and chemical industries.

Keywords: SGNH hydrolase; Est24; Immobilization; Industrial applications

Identification of a brevianamide F reverse prenyltransferase BrePT from Aspergillus versicolor with a broad substrate specificity towards tryptophan-containing cyclic dipeptides by Suqin Yin; Xia Yu; Qing Wang; Xiao-Qing Liu; Shu-Ming Li (pp. 1649-1660).

A putative brevianamide F reverse prenyltransferase gene brePT was amplified from Aspergillus versicolor NRRL573 by using primers deduced from its orthologue notF in Aspergillus sp. MF297-2 and overexpressed in Escherichia coli. The soluble His-tagged protein BrePT was purified to near homogeneity and assayed with tryptophan-containing cyclic dipeptides in the presence of dimethylallyl diphosphate. BrePT showed much higher flexibility towards its aromatic substrates than NotF and accepted all of the 14 tested tryptophan-containing cyclic dipeptides. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific reverse prenylation at C2 of the indole nucleus. K _M values of BrePT were determined for its putative substrates brevianamide F and DMAPP at 32 and 98 μM, respectively. Average turnover number (k _cat) at 0.4 s⁻¹ was calculated from kinetic data of brevianamide F and DMAPP. K _M values in the range of 0.082–2.9 mM and k _cat values from 0.003 to 0.15 s⁻¹ were determined for other 11 cyclic dipeptides. Similar to known fungal indole prenyltransferases, BrePT did not accept geranyl or farnesyl diphosphate as prenyl donor for its prenylation.

Keywords: Brevianamide; Cyclic dipeptide; DMATS superfamily; Prenylated derivative; Prenyltransferase

Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes by Stéphane Pesce; Jérémie Beguet; Nadine Rouard; Marion Devers-Lamrani; Fabrice Martin-Laurent (pp. 1661-1668).

A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes’ (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-¹⁴C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sediment communities, mineralization potential did not depend solely on the quantity of puhB copies, underlining the need to assess gene expression. In the sediment samples, both puhB copy numbers and mineralization capacities were highly conditioned by whether or not diuron-treated soil was added. This points to transfers of degradative potential from soils to sediments. No puhA gene was detected in soil and sediment DNA extracts. Moreover, some sediments exhibited high diuron mineralization potential even though puhB genes were not detected, suggesting the existence of alternative diuron degradation pathways.

Keywords: Biodegradation; Microbial communities; Mineralization; Pesticides; Phenylurea hydrolases; qPCR

Implementation of a transhydrogenase-like shunt to counter redox imbalance during xylose fermentation in Saccharomyces cerevisiae by Hiroyuki Suga; Fumio Matsuda; Tomohisa Hasunuma; Jun Ishii; Akihiko Kondo (pp. 1669-1678).

Three enzymes responsible for the transhydrogenase-like shunt, including malic enzyme (encoded by MAE1), malate dehydrogenase (MDH2), and pyruvate carboxylase (PYC2), were overexpressed to regulate the redox state in xylose-fermenting recombinant Saccharomyces cerevisiae. The YPH499XU/MAE1 strain was constructed by overexpressing native Mae1p in the YPH499XU strain expressing xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and native xylulokinase. Analysis of the xylose fermentation profile under semi-anaerobic conditions revealed that the ethanol yield in the YPH499XU/MAE1 strain (0.38 ± 0.01 g g⁻¹ xylose consumed) was improved from that of the control strain (0.31 ± 0.01 g g⁻¹ xylose consumed). Reduced xylitol production was also observed in YPH499XU/MAE1, suggesting that the redox balance was altered by Mae1p overexpression. Analysis of intracellular metabolites showed that the redox imbalance during xylose fermentation was partly relieved in the transformant. The specific ethanol production rate in the YPH499XU/MAE1–MDH2 strain was 1.25-fold higher than that of YPH499XU/MAE1 due to the additional overexpression of Mdh2p, whereas the ethanol yield was identical to that of YPH499XU/MAE1. The specific xylose consumption rate was drastically increased in the YPH499XU/MAE1–MDH2–PYC2 strain. However, poor ethanol yield as well as increased production of xylitol was observed. These results demonstrate that the transhydrogenase function implemented in S. cerevisiae can regulate the redox state of yeast cells.

Keywords: Transhydrogenase-like shunt; Malic enzyme; Anaplerotic pathway; Xylose fermentation; Saccharomyces cerevisiae

Glucosamine as carbon source for amino acid-producing Corynebacterium glutamicum by Andreas Uhde; Jung-Won Youn; Tomoya Maeda; Lina Clermont; Christian Matano; Reinhard Krämer; Volker F. Wendisch; Gerd M. Seibold; Kay Marin (pp. 1679-1687).

Corynebacterium glutamicum grows with a variety of carbohydrates and carbohydrate derivatives as sole carbon sources; however, growth with glucosamine has not yet been reported. We isolated a spontaneous mutant (M4) which is able to grow as fast with glucosamine as with glucose as sole carbon source. Glucosamine also served as a combined source of carbon, energy and nitrogen for the mutant strain. Characterisation of the M4 mutant revealed a significantly increased expression of the nagB gene encoding the glucosamine-6P deaminase NagB involved in degradation of glucosamine, as a consequence of a single mutation in the promoter region of the nagAB-scrB operon. Ectopic nagB overexpression verified that the activity of the NagB enzyme is in fact the growth limiting factor under these conditions. In addition, glucosamine uptake was studied, which proved to be unchanged in the wild-type and M4 mutant strains. Using specific deletion strains, we identified the PTS^Glc transport system to be responsible for glucosamine uptake in C. glutamicum. The affinity of this uptake system for glucosamine was about 40-fold lower than that for its major substrate glucose. Because of this difference in affinity, glucosamine is efficiently taken up only if external glucose is absent or present at low concentrations. C. glutamicum was also examined for its suitability to use glucosamine as substrate for biotechnological purposes. Upon overexpression of the nagB gene in suitable C. glutamicum producer strains, efficient production of both the amino acid l-lysine and the diamine putrescine from glucosamine was demonstrated.

Keywords: Corynebacterium glutamicum ; Glucosamine; NagB; PtsG; l-Lysine; Putrescine

Alterations of the gut microbiota in high-fat diet mice is strongly linked to oxidative stress by Yi Qiao; Jin Sun; Yinyi Ding; Guowei Le; Yonghui Shi (pp. 1689-1697).

Alterations of the gut microbiota induced by diet exert a strong influence on the development of metabolic syndrome. In this study, we prove the hypothesis that the long-term high-fat diet (HFD) may influence gut microbiota directly and/or indirectly by changing the redox state. Lipoic acid (LA), as a universal antioxidant, was used to improve the redox state. Reactive oxygen species (ROS), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were analyzed to profile oxidative stress states. PCR-denaturing gradient gel electrophoresis (DGGE) was used to describe gut flora structures, while plate count was employed for the quantitative analysis of Escherichia coli, lactobacilli, and enterococcus. The influence of redox state on the vitality of gut-derived bacteria was measured in vitro. ROS and MDA, which significantly decreased in LA mice compared with HFD mice, showed a strong positive association with E. coli and enterococcus (P < 0.05) and a negative association with lactobacilli (P < 0.05). Increased T-AOC in LA mice showed a high positive association with lactobacilli (P < 0.05) and a negative correlation with E. coli and enterococcus. These correlations implied that the dietary effects on the gut microbiota were conferred, at least in part, through an effect on oxidative stress. This study provides evidence that modulation of the redox state by an antioxidant has the potential to improve gut microbiota, which has relevance for metabolic health.

Keywords: Gut microbiota; Oxidative stress; High-fat diet; Lipoic acid

Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production by Odile Francesca Restaino; Irene di Lauro; Donatella Cimini; Elisabetta Carlino; Mario De Rosa; Chiara Schiraldi (pp. 1699-1709).

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological–chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.

Keywords: Chondroitin sulphate; Escherichia coli K4; Capsular polysaccharide precursor; K4 polymerase

Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes by Lirong Li; YongHui Shi; Maureen Jepkorir Cheserek; GuanFang Su; GuoWei Le (pp. 1711-1723).

A number of research have proven that antimicrobial peptides are of greatest potential as a new class of antibiotics. Antimicrobial peptides and cell-penetrating peptides share some similar structure characteristics. In our study, a new peptide analog, APP (GLARALTRLLRQLTRQLTRA) from the cell-penetrating peptide ppTG20 (GLFRALLRLLRSLWRLLLRA), was identified simultaneously with the antibacterial mechanism of APP against Salmonella typhimurium and Streptococcus pyogenes. APP displayed potent antibacterial activity against Gram-negative and Gram-positive strains. The minimum inhibitory concentration was in the range of 2 to 4 μM. APP displayed higher cell selectivity (about 42-fold increase) as compared to the parent peptide for it decreased hemolytic activity and increased antimicrobial activity. The calcein leakage from egg yolk l-α-phosphatidylcholine (EYPC)/egg yolk l-α-phosphatidyl-dl-glycerol and EYPC/cholesterol vesicles demonstrated that APP exhibited high selectivity. The antibacterial mechanism analysis indicated that APP induced membrane permeabilization in a kinetic manner for membrane lesions allowing O-nitrophenyl-β-d-galactoside uptake into cells and potassium release from APP-treated cells. Flow cytometry analysis demonstrated that APP induced bacterial live cell membrane damage. Circular dichroism, fluorescence spectra, and gel retardation analysis confirmed that APP interacted with DNA and intercalated into the DNA base pairs after penetrating the cell membrane. Cell cycle assay showed that APP affected DNA synthesis in the cell. Our results suggested that peptides derived from the cell-penetrating peptide have the potential for antimicrobial agent development, and APP exerts its antibacterial activity by damaging bacterial cell membranes and binding to bacterial DNA to inhibit cellular functions, ultimately leading to cell death.

Keywords: Antibacterial peptide; Antimicrobial activity; Cell-penetrating peptide; DNA-binding activity

Zinc oxide-doped poly(urethane) spider web nanofibrous scaffold via one-step electrospinning: a novel matrix for tissue engineering by Touseef Amna; M. Shamshi Hassan; Faheem A. Sheikh; Hak Kyo Lee; Kang-Seok Seo; Duhak Yoon; I. H. Hwang (pp. 1725-1734).

Zinc oxide (ZnO) nanostructures have been commonly studied for electronic purposes due to their unique piezoelectric and catalytic properties; however, recently, they have been also exploited for biomedical applications. The purpose of this study was to fabricate ZnO-doped poly(urethane) (PU) nanocomposite via one-step electrospinning technique. The utilized nanocomposite was prepared by using colloidal gel composed of ZnO and PU, and the obtained mats were vacuum dried at 60 °C overnight. The physicochemical characterization of as-spun composite nanofibers was carried out by X-ray diffraction pattern, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, electron probe microanalysis, and transmission electron microscopy, whereas the thermal behavior was analyzed by thermogravimetric analysis. The viability, attachment, and proliferation of NIH 3T3 mouse fibroblast cells on the ZnO/PU composite nanofibers were analyzed by in vitro cell compatibility test. The morphological features of the cells attached on nanofibers were examined by Bio-SEM. We conclude that the electrospun nanofibrous scaffolds with unique spider nets had good biocompatibility. Cytotoxicity experiments indicated that the mouse fibroblasts could attach to the nanocomposite after being cultured. Thus, the current work demonstrates that the as-synthesized ZnO/PU hybrid nanofibers represent a promising biomaterial to be exploited for various tissue engineering applications.

Keywords: Electrospinning; Composite nanofibers; NIH 3T3 fibroblasts; Cytotoxicity; Spider net

Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains by Juan Daniel Rivaldi; Marta Luís C. Sousa Silva; Luis C. Duarte; António E. N. Ferreira; Carlos Cordeiro; Maria das Graças de Almeida Felipe; Ana de Ponces Freire; Ismael Maciel de Mancilha (pp. 1735-1743).

Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h. L. acidophilus and L. delbrueckii showed a similar growth, higher than L. plantarum. The highest biomass reached was 2.11 g L⁻¹ for L. acidophilus, with a cell mass yield (Y _X/S) of 0.37 g g⁻¹. L. delbrueckii and L. plantarum reached a biomass of 2.06 and 1.36 g L⁻¹. All strains catabolize glycerol mainly through glycerol kinase (EC 2.7.1.30). For these lactobacillus species, kinetic parameters for glycerol kinase showed Michaelis–Menten constant (K _m) ranging from 1.2 to 3.8 mM. The specific activities for glycerol kinase in these strains were in the range of 0.18 to 0.58 U mg protein⁻¹, with L. acidophilus ATCC 4356 showing the maximum specific activity after 24 h of cultivation. Glycerol dehydrogenase activity was also detected in all strains studied but only for the reduction of glyceraldehyde with NADPH (K _m for DL-glyceraldehyde ranging from 12.8 to 32.3 mM). This enzyme shows a very low oxidative activity with glycerol and NADP⁺ and, most likely, under physiological conditions, the oxidative reaction does not occur, supporting the assumption that the main metabolic flux concerning glycerol metabolism is through the glycerol kinase pathway.

Keywords: Biodiesel-derived glycerol; Glycerol kinase; Glycerol dehydrogenase; Lactobacillus ; Enzyme kinetics; Biomass production

Effect of COD/N ratio on cultivation of aerobic granular sludge in a pilot-scale sequencing batch reactor by Dong Wei; Zhuangming Qiao; Yongfang Zhang; Lianjie Hao; Wei Si; Bin Du; Qin Wei (pp. 1745-1753).

Aerobic granular sludge was successfully cultivated with the effluent of internal circulation reactor in a pilot-scale sequencing batch reactor (SBR). Soy protein wastewater was used as an external carbon source for altering the influent chemical oxygen demand/nitrogen (COD/N) ratios of SBR. Initially, the phenomenon of partial nitrification was observed and depressed by increasing the influent COD/N ratios from 3.32 to 7.24 mg/mg. After 90 days of aerobic granulation, the mixed liquor suspended solids concentration of the reactor increased from 2.80 to 7.02 g/L, while the sludge volumetric index decreased from 105.51 to 42.99 mL/g. The diameters of mature aerobic granules vary in the range of 1.2 to 2.0 mm. The reactor showed excellent removal performances for COD and $$ { ext{NH}}_4^{ + }{ ext{ - N}} $$ after aerobic granulation, and average removal efficiencies were over 93% and 98%, respectively. The result of this study could provide further information on the development of aerobic granule-based system for full-scale applications.

Keywords: Aerobic granular sludge; Pilot-scale; COD/N ratio; Sequencing batch reactor (SBR)

General and rare bacterial taxa demonstrating different temporal dynamic patterns in an activated sludge bioreactor by Taek-Seung Kim; Ju-Yong Jeong; George F. Wells; Hee-Deung Park (pp. 1755-1765).

Temporal variation of general and rare bacterial taxa was investigated using pyrosequencing of 16S rRNA gene from activated sludge samples collected bimonthly for a two-year period. Most of operational taxonomic units (OTUs) were allocated to rare taxa (89.6%), but the rare taxa comprised a small portion of the community in terms of abundance of sequences analyzed (28.6%). Temporal variations in OTUs richness significantly differed between the two taxa groups in which the rare taxa showed a higher diversity and a more fluctuating pattern than the general taxa. Furthermore, the two taxa groups were constrained by different explanatory variables: influent BOD, effluent BOD, and DO were the significant (P < 0.05) parameters affecting the pattern of the general taxa, while temperature was the factor for the rare taxa. Over the test period, the general taxa persisted for a longer time (i.e., lower turnover rate) in the bioreactor than the rare taxa. In conclusion, this study demonstrated clear differences in temporal dynamic patterns for the general and rare bacterial taxa in an activated sludge bioreactor, which would be a foundation for better understanding the bacterial ecology of activated sludge.

Keywords: Activated sludge; Rare species; Pyrosequencing; Species time relationship; Temporal dynamics

Bioenergetics for the growth of Staphylococcus lentus in biocompatible choline salts by Sudharshan Sekar; Surianarayanan Mahadevan; Ranganathan Vijayaraghavan; Asit Baran Mandal; D. R. MacFarlane (pp. 1767-1774).

Choline-based biocompatible salts were used as “nutrients” for the growth of Staphylococcus lentus bacteria. Increase in the growth rate of bacteria was observed, compared to conventional carbon sources. In the case of the ionic liquid, choline lactate, the increase was pronounced. Bacterial growth was correlated with power–time curve in an investigation monitored online by reaction calorimetry. From the power–time curve, three phases of the growth can be distinctly seen. Heat yield coefficients estimated for the growth of S. lentus were found to match well with those reported hitherto. A comparative study of heat yields (catabolic) between glucose and choline lactate revealed significant information; the heat yield due to choline lactate (Y _Q/S) consumption and oxygen (Y _Q/O) were 23.4 kJ/g and 435 kJ/mol and whereas that for glucose with oxygen were 9.6 kJ/g and 427 kJ/mol, respectively, showing clearly the preferential affinity of choline lactate by the bacteria rather than glucose. This study also established that the use of ionic liquids as nutrients can be monitored using bioreaction calorimetry.

Keywords: Staphylococcus lentus ; Bioenergetics; Biological reaction calorimetry; Choline lactate; Ionic liquid

Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica) by Ting Wei; Jun Hu; Kazuhiko Miyanaga; Yasunori Tanji (pp. 1775-1783).

The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR−DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.

Keywords: Housefly; Bacterial association; Multidrug resistance; Vector potential

Dominance of Candidatus Scalindua species in anammox community revealed in soils with different duration of rice paddy cultivation in Northeast China by Jing Wang; Ji-Dong Gu (pp. 1785-1798).

The anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the oxygen-limited zone for nitrogen cycling, but their roles in agricultural ecosystems are still poorly understood. In this study, soil samples were taken from the rhizosphere and non-rhizosphere and from surface (0–5 cm) and subsurface (20–25 cm) layers with 1, 4, and 9 years of rice cultivation history on the typical albic soil of Northeast China to examine the diversity and distribution of anammox bacteria based on 16S rRNA gene and hydrazine oxidoreductase encoding gene (hzo). By comparing these soil samples, no obvious difference was observed in community composition between the rhizosphere and non-rhizosphere or the surface and subsurface layers. Surprisingly, anammox bacterial communities of these rice paddy soils were consisted of mainly Candidatus Scalindua species, which are best known to be dominant in marine and pristine environments. The highest diversity was revealed in the 4-year paddy soil based on clone library analysis. Phylogenetic analysis of 16S rRNA gene and deduced HZO from the corresponding encoding gene showed that most of the obtained clones are grouped together with Candidatus Scalindua sorokinii, Candidatus Scalindua brodae, and Candidatus Scalindua spp. of seawater. The obtained clone sequences from all samples are distributed in two subclusters that contain sequences from environmental samples only. Tentative new species were also discovered in this paddy soil. This study provides the first evidence on the existence of anammox bacteria with limited diversity in agricultural ecosystems in Northern China.

Keywords: Anammox bacteria; Rice paddy soil; 16S rRNA gene; Hydrazine oxidoreductase gene

Phytoremediation of triphenylmethane dyes by overexpressing a Citrobacter sp. triphenylmethane reductase in transgenic Arabidopsis by Xiao-Yan Fu; Wei Zhao; Ai-Sheng Xiong; Yong-Sheng Tian; Bo Zhu; Ri-He Peng; Quan-Hong Yao (pp. 1799-1806).

Triphenylmethane dyes are extensively utilized in textile industries, medicinal products, biological stains, and food processing industries, etc. They are generally considered as xenobiotic compounds, which are very recalcitrant to biodegradation. The widespread persistence of such compounds has generated concerns with regard to remediation of them because of their potential carcinogenicity, teratogenicity, and mutagenicity. In this study, we present a system of phytoremediation by Arabidopsis plants developed on the basis of overexpression of triphenylmethane reductase (TMR) from the Citrobacter sp. The morphology and growth of TMR transgenic Arabidopsis plants showed significantly enhanced tolerances to crystal violet (CV) and malachite green (MG). Further, HPLC and HPLC–MS analyses of samples before and after dye decolorization in culture media revealed that TMR transgenic plants exhibited strikingly higher capabilities of removing CV from their media and high efficiencies of converting CV to non-toxic leucocrystal violet (LCV). This work indicates that microbial degradative gene may be transgenically exploited in plants for bioremediation of triphenylmethane dyes in the environment.

Keywords: Phytoremediation; Citrobacter sp.; Triphenylmethane reductase; Triphenylmethane dyes; Transgenic Arabidopsis

Erratum to: Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains by Juan Daniel Rivaldi; Marta Sousa Silva; Luis C. Duarte; António E. N. Ferreira; Carlos Cordeiro; Maria das Graças de Almeida Felipe; Ana Ponces Freire; Ismael Maciel de Mancilha (pp. 1807-1807).

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Applied Microbiology and Biotechnology (v.97, #4)

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Applied Microbiology and Biotechnology (v.97, #4)