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Applied Microbiology and Biotechnology (v.95, #2)

Metallic oxide nanoparticles: state of the art in biogenic syntheses and their mechanisms by Nelson Durán; Amedea B. Seabra (pp. 275-288).

This review presents the syntheses and characterizations of size and morphology, as well as the mechanistic aspects, of metallic oxide nanoparticles synthesized by biogenic processes. Furthermore, the importance of their biogenic synthesis is compared with chemical synthesis, and their applications are discussed from the ecological and environmental view points. To our best knowledge, this review presents for the first time the synthesis of several biogenic oxide nanoparticles, with great applications under the perspective of cost effective and eco-friendly points of view.

Keywords: Metallic oxide nanoparticles; Biogenic synthesis; Biosynthesis; Nanobiotechnology

Capability of yeast derivatives to adhere enteropathogenic bacteria and to modulate cells of the innate immune system by Anja Ganner; Gerd Schatzmayr (pp. 289-297).

Yeast derivatives including yeast cell wall components are promising alternatives to antibiotics with respect to the promotion of health and performance in livestock, based on their capacity to bind enteropathogenic bacteria and to beneficially modulate the immune system. However, these mode(s) of action both in vitro and in vivo are still not well understood. Furthermore, standardization and reproducibility of in vitro techniques (microbiology, cell culture assays) are critical features for the application of yeast derivatives as well as for the proof of effectiveness. Yeast cell wall products are suggested as anti-adhesive agents and are thus proposed to prevent attachment of certain intestinal bacteria by providing alternative adhesion sites to enterobacteria, which contain mannose-specific type I fimbriae such as Escherichia coli or Salmonella spp. and which is well documented. Various in vitro assay techniques have become of paramount importance for biotechnological research since they allow for determination and quantification of potential mode(s) of action. However, in vitro assays may be criticized by product end users as not accurately reflecting in vivo responses. Pro and cons of different assays and their bias will be discussed specifically regarding yeast cell wall components and adhesion of enteropathogenic bacteria. Immunomodulation is a therapeutic approach intervening in auto-regulating processes of the defense system. Yeast derivatives such as beta-glucans are proposed to interact with cells of the innate immune system by receptor recognition. Controversial data in literature and mode(s) of action are reviewed and discussed here.

Keywords: Yeast derivatives; Adhesion; Enteropathogenic bacteria; Immunomodulation; In vitro assays; Cytokines

Efficient surface modification of biomaterial to prevent biofilm formation and the attachment of microorganisms by Kateryna Bazaka; Mohan V. Jacob; Russell J. Crawford; Elena P. Ivanova (pp. 299-311).

Biomaterials play a fundamental role in disease management and the improvement of health care. In recent years, there has been a significant growth in the diversity, function, and number of biomaterials used worldwide. Yet, attachment of pathogenic microorganisms onto biomaterial surfaces remains a significant challenge that substantially undermines their clinical applicability, limiting the advancement of these systems. The emergence and escalating pervasiveness of antibiotic-resistant bacterial strains makes the management of biomaterial-associated nosocomial infections increasingly difficult. The conventional post-operative treatment of implant-caused infections using systemic antibiotics is often marginally effective, further accelerating the extent of antimicrobial resistance. Methods by which the initial stages of bacterial attachment and biofilm formation can be restricted or prevented are therefore sought. The surface modification of biomaterials has the potential to alleviate pathogenic biofouling, therefore preventing the need for conventional antibiotics to be applied.

Keywords: Biomaterials; Surface treatment; Biofilm; Bacterial attachment

Functions, applications and production of 2-O-d-glucopyranosyl-l-ascorbic acid by Ruizhi Han; Long Liu; Jianghua Li; Guocheng Du; Jian Chen (pp. 313-320).

Vitamin C (VC) is an essential nutrient that cannot be synthesized by the human body. Due to its extreme instability, various VC derivatives have been developed in an attempt to improve stability while retaining the same biological activity. One of the most important VC derivatives, 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G), has attracted increasing attention in recent years with a wide range of applications in cosmetics, food, and medicine. In this mini-review, we first introduce the types and properties of different VC glycosyl derivatives. Next, we provide an overview of the functions and applications of AA-2G. Finally, we discuss in-depth the current status and future prospects of AA-2G production by biotransformation.

Keywords: l-ascorbic acid; 2-O-d-glucopyranosyl-l-ascorbic acid; Applications; Biotransformation

Production of biogas from solid organic wastes through anaerobic digestion: a review by Ismail Muhammad Nasir; Tinia I. Mohd Ghazi; Rozita Omar (pp. 321-329).

Anaerobic digestion treatments have often been used for biological stabilization of solid wastes. These treatment processes generate biogas which can be used as a renewable energy sources. Recently, anaerobic digestion of solid wastes has attracted more interest because of current environmental problems, most especially those concerned with global warming. Thus, laboratory-scale research on this area has increased significantly. In this review paper, the summary of the most recent research activities covering production of biogas from solid wastes according to its origin via various anaerobic technologies was presented.

Keywords: Anaerobic digestion; Biogas; Methane; Solid waste

Impact of Azotobacter exopolysaccharides on sustainable agriculture by Samiran S. Gauri; Santi M. Mandal; Bikas R. Pati (pp. 331-338).

Recently, increasing attention have lead to search other avenue of biofertilizers with multipurpose activities as a manner of sustainable soil health to improve the plant productivity. Azotobacter have been universally accepted as a major inoculum used in biofertilizer to restore the nitrogen level into cultivated field. Azotobacter is well characterized for their profuse production of exopolysaccharides (EPS). Several reviews on biogenesis and multifunctional role of Azotobacter EPS have been documented with special emphasis on industrial applications. But the impact of Azotobacter EPS in plant growth promotion has not received adequate attention. This review outlines the evidence that demonstrates not only the contribution of Azotobacter EPS in global nutrient cycle but also help to compete successfully in different adverse ecological and edaphic conditions. This also focuses on new insights and concepts of Azotobacter EPS which have positive effects caused by the biofilm formation on overall plant growth promotion with other PGPRs. In addition, their potentials in agricultural improvement are also discussed. Recent data realized that Azotobacter EPS have an immense agro-economical importance including the survivability and maintenance of microbial community in their habitat. This leads us to confirm that the next generation Azotobacter inoculum with high yielding EPS and high nitrogen fixing ability can be utilized to satisfy the future demand of augmented crop production attributed to increase plant growth promoting agents.

Keywords: Azotobacter ; Biofertilizer; Biofilm formation; Ecological significance; Exopolysaccharides

Aspergillus and Penicillium identification using DNA sequences: barcode or MLST? by Stephen W. Peterson (pp. 339-344).

Current methods in DNA technology can detect single nucleotide polymorphisms with measurable accuracy using several different approaches appropriate for different uses. If there are even single nucleotide differences that are invariant markers of the species, we can accomplish identification through rapid DNA-based tests. The question of whether we can reliably detect and identify species of Aspergillus and Penicillium turns mainly upon the completeness of our alpha taxonomy, our species concepts, and how well the available DNA data coincide with the taxonomic diversity in the family Trichocomaceae. No single gene is yet known that is invariant within species and variable between species as would be optimal for the barcode approach. Data are published that would make an MLST approach to isolate identification possible in the most well-studied clades of Aspergillus and Penicillium.

Keywords: Barcode; MLST; Species concept; Trichocomaceae

Flow cytometric assessment of the protectants for enhanced in vitro survival of probiotic lactic acid bacteria through simulated human gastro-intestinal stresses by Song Chen; Yu Cao; Lynnette R. Ferguson; Quan Shu; Sanjay Garg (pp. 345-356).

The aim of this study was to apply flow cytometric (FCM) analysis to assess the use of sucrose and lecithin vesicles for the protection of probiotic lactic acid bacteria in response to the challenge of gastric acidity and bile salts. FCM analysis in combination with fluorescent probes carboxyfluorescein (cF) and propidium iodide was used to reveal the physiological heterogeneity in the stressed bacteria population. Three subpopulations (intact, stressed, and damaged) were differentiated by FCM in all six examined strains. Significant changes were observed in the presence of the selected protectants. The addition of 20 mM sucrose in the simulated gastric fluid substantially increased the number of intact cells over 20 folds and reduced the damaged subpopulation by half. The presence of 2 % (w/v) lecithin vesicles was shown to protect 50 % more intact cells from the challenge of bile salts. The improved survival as evaluated by FCM analysis was further assessed for the proliferation capacity by sorting a number of cells from each subpopulation on nutrient agar plate. The result confirmed conformity between the proliferation-based cultivability and the probe-indicated viability in the samples of the intact and the damaged subpopulations. However, it also revealed the complexities of the stressed (injured) subpopulation. In conclusion, FCM analysis confirmed that the selected protectants could improve the survival of the probiotic strains in the simulated GI environments. The FCM analysis also proved to be a useful analytical tool for the probiotics research.

Keywords: Probiotics; Flow cytometry; Metabolisable sugar; Lecithin vesicle; Bile salt; Gastric pH; Lactic acid bacteria; Cell sorting

Development of a whole-cell biocatalyst co-expressing P450 monooxygenase and glucose dehydrogenase for synthesis of epoxyhexane by Akasit Siriphongphaew; Pimpaya Pisnupong; Jirarut Wongkongkatep; Pranee Inprakhon; Alisa S. Vangnai; Kohsuke Honda; Hisao Ohtake; Junichi Kato; Jun Ogawa; Sakayu Shimizu; Vlada B. Urlacher; Rolf D. Schmid; Thunyarat Pongtharangkul (pp. 357-367).

Oxygenases-based Escherichia coli whole-cell biocatalyst can be applied for catalysis of various commercially interesting reactions that are difficult to achieve with traditional chemical catalysts. However, substrates and products of interest are often toxic to E. coli, causing a disruption of cell membrane. Therefore, organic solvent-tolerant bacteria became an important tool for heterologous expression of such oxygenases. In this study, the organic solvent-tolerant Bacillus subtilis 3C5N was developed as a whole-cell biocatalyst for epoxidation of a toxic terminal alkene, 1-hexene. Comparing to other hosts tested, high level of tolerance towards 1-hexene and a moderately hydrophobic cell surface of B. subtilis 3C5N were suggested to contribute to its higher 1,2-epoxyhexane production. A systematic optimization of reaction conditions such as biocatalyst and substrate concentration resulted in a 3.3-fold increase in the specific rate. Co-expression of glucose dehydrogenase could partly restored NADPH-regenerating ability of the biocatalyst (up to 38 % of the wild type), resulting in approximately 53 % increase in specific rate representing approximately 22-fold increase in product concentration comparing to that obtained prior to an optimization.

Keywords: P450 BM3; Bacillus subtilis ; Epoxidation; Whole-cell biocatalyst

Alternaria sp. MG1, a resveratrol-producing fungus: isolation, identification, and optimal cultivation conditions for resveratrol production by Junling Shi; Qin Zeng; Yanlin Liu; Zhongli Pan (pp. 369-379).

Due to its potential in preventing or slowing the occurrence of many diseases, resveratrol (3,5,4′-trihydroxystilbene) has attracted great research interest. The objective of this study was to identify microorganisms from selected plants that produce resveratrol and to optimize the conditions for resveratrol production. Endophytes from Merlot wine grapes (Vitis vinifera L. cv. Merlot), wild Vitis (Vitis quinquangularis Rehd.), and Japanese knotweed (Polygonum cuspidatum Siebold & Zucc.) were isolated, and their abilities to produce resveratrol were evaluated. A total of 65 isolates were obtained and 21 produced resveratrol (6–123 μg/L) in liquid culture. The resveratrol-producing isolates belonged to seven genera, Botryosphaeria, Penicillium, Cephalosporium, Aspergillus, Geotrichum, Mucor, and Alternaria. The resveratrol-producing capability decreased or was completely lost in most isolates after three rounds of subculture. It was found that only the strain Alternaria sp. MG1 (isolated from cob of Merlot using GA1 medium) had stable and high resveratrol-producing capability in all subcultures. During liquid cultivation of Alternaria sp. MG1 in potato dextrose medium, the synthesis of resveratrol began on the first day, increased to peak levels on day 7, and then decreased sharply thereafter. Cell growth increased during cultivation and reached a stable and high level of biomass after 5 days. The best fermentation conditions for resveratrol production in liquid cultures of Alternaria sp. MG1 were an inoculum size of 6 %, a medium volume of 125 mL in a 250-mL flask, a rotation speed of 101 rpm, and a temperature of 27 °C.

Keywords: Resveratrol; Endophyte; Alternaria ; Response surface method

Selective biosorption and recovery of Ruthenium from industrial effluents with Rhodopseudomonas palustris strains by Giovanni Colica; Stefania Caparrotta; Roberto De Philippis (pp. 381-387).

This study demonstrated for the first time the possibility to remove and partially recover the Ruthenium contained in industrial effluents by using purple non sulfur bacteria (PNSB) as microbial biosorbents. Up to date, the biosorption was only claimed as possible tool for the removal of the platinum-group metals (PGM) but the biosorption of Ru was never experimentally investigated. The PNSBs tested have adsorbed around 40 mg g (dry biomass)⁻¹ of the Ru contained in the real industrial effluents. At the end of the bioremoval experiments, the amount of Ru recovered from the biomass ranged from 42 % to 72 % of that adsorbed by PNSB, depending by the characteristics of the Ru effluent used. In any case, the use of microbial sorbents such as PNSB for the biosorption and recovery of Ru can be considered a way to reduce both the costs and the impact on the environment of the mining activities needed to obtain the increasing amounts of this rare and precious metal requested by the industrial activities related to its use.

Keywords: Ruthenium; Biosorption; Metal recovery; Rhodopseudomonas palustris ; Electroplating; Industrial effluents

Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B by Remko T. Winter; Dominic P. H. M. Heuts; Egon M. A. Rijpkema; Edwin van Bloois; Hein J. Wijma; Marco W. Fraaije (pp. 389-403).

We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene (sharing 48% protein sequence identity to AldO) was identified, cloned and expressed in Escherichia coli. Following 6xHis tag purification, characterization revealed the protein to be a covalent flavoprotein of 47 kDa with a remarkably similar reactivity and substrate specificity to that of AldO. A steady-state kinetic analysis with a number of different polyol substrates revealed lower catalytic rates but slightly altered substrate specificity when compared to AldO. Thermostability measurements revealed that the novel AldO is a highly thermostable enzyme with an unfolding temperature of 84 °C and an activity half-life at 75 °C of 112 min, prompting the name HotAldO. Inspired by earlier studies, we attempted a straightforward, exploratory approach to improve the thermostability of AldO by replacing residues with high B-factors with corresponding residues from HotAldO. None of these mutations resulted in a more thermostable oxidase; a fact that was corroborated by in silico analysis.

Keywords: Carbohydrate oxidase; Flavoenzyme; Thermostable; ThermoFAD; Alditols

Directed evolution of a highly active Yersinia mollaretii phytase by Amol V. Shivange; Annegret Serwe; Alexander Dennig; Danilo Roccatano; Stefan Haefner; Ulrich Schwaneberg (pp. 405-418).

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073 U/mg which is ∼10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ∼8,400 clones) a phytase variant with ∼20% improved thermostability (58°C for 20 min; residual activity wild type ∼34%; variant ∼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993 U/mg).

Keywords: Directed evolution; High-throughput screening; SeSaM; Phytase; Thermostability

Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119 by Uschara Thumarat; Ryota Nakamura; Takeshi Kawabata; Hideyuki Suzuki; Fusako Kawai (pp. 419-430).

Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS–PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca²⁺ and was stable up to 60 °C with 150 mM Ca²⁺. Another identical gene was located in tandem in the upstream of est119.

Keywords: Polyester degradation; Thermobifida alba AHK119; Cutinase-type polyesterase; Ca-dependent cutinase; Thermostable polyesterase

l-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of l-pantoyl lactone by Dayong Si; Nobuyuki Urano; Shinya Nozaki; Kohsuke Honda; Sakayu Shimizu; Michihiko Kataoka (pp. 431-440).

The 1,2-propanediol (1,2-PD) inducible membrane-bound l-pantoyl lactone (l-PL) dehydrogenase (LPLDH) has been isolated from Rhodococcus erythropolis AKU2103 (Kataoka et al. in Eur J Biochem 204:799, 1992). Based on the N-terminal amino acid sequence of LPLDH and the highly conserved amino acid sequence in homology search results, the LPLDH gene (lpldh) was cloned. The gene consists of 1,179 bases and encodes a protein of 392 amino acid residues. The deduced amino acid sequence showed high similarity to the proteins of the FMN-dependent α-hydroxy acid dehydrogenase/oxidase family. The overexpression vector pKLPLDH containing lpldh with its upstream region (1,940 bp) was constructed and introduced into R. erythropolis AKU2103. The recombinant R. erythropolis AKU2103 harboring pKLPLDH showed six times higher LPLDH activity than the wild-type strain. Conversion of l-PL to ketopantoyl lactone was achieved with 92% or 80% conversion yield when the substrate concentration was 0.768 or 1.15 M, respectively. Stereoinversion of l-PL to d-PL was also carried out by using the combination of recombinant R. erythropolis AKU2103 harboring pKLPLDH and ketopantoic acid-reducing Escherichia coli.

Keywords: l-Pantoyl lactone dehydrogenase; Rhodococcus erythropolis ; Ketopantoyl lactone; Stereoinversion

Promotion of glycerol utilization using ethanol and 1-propanol in Schizosaccharomyces pombe by Tomohiko Matsuzawa; Futoshi Hara; Hideki Tohda; Hiroshi Uemura; Kaoru Takegawa (pp. 441-449).

The fission yeast Schizosaccharomyces pombe does not grow in media containing glycerol as a sole carbon source but uses glycerol in the presence of ethanol. Ethanol, but not glycerol, triggered upregulation of gld1 ⁺ and fbp1 ⁺ during glucose starvation even though gld1 ⁺ and fbp1 ⁺ are essential for growth on glycerol. This upregulation occurred at a very low concentration of ethanol. The transcriptional regulation of gld1 ⁺ was tested in the presence of various alcohols, and both ethanol and 1-propanol were found to induce gld1 ⁺ and to support growth in glycerol-containing media. We suggest that S. pombe has a novel ethanol and/or 1-propanol recognition mechanism that upregulates glycerol utilization during glucose starvation.

Keywords: Schizosaccharomyces pombe ; Glycerol; Ethanol; Transcriptional activation

Molecular cloning of the gene cluster for lariatin biosynthesis of Rhodococcus jostii K01-B0171 by Junji Inokoshi; Maki Matsuhama; Midori Miyake; Haruo Ikeda; Hiroshi Tomoda (pp. 451-460).

The biosynthetic gene cluster for lariatins A and B, anti-mycobacterial peptide antibiotics with a unique “lasso” structure, was cloned from Gram-positive bacterium Rhodococcus jostii K01-B0171. Random transposition mutagenesis using IS1415 derivative was carried out to identify a chromosomal locus involved in lariatin biosynthesis and six independent lariatin non-producing variants were obtained. Arbitrary PCR revealed that one insertion was located near the region involved in lariatin biosynthesis. Using the lariatin gene as a probe, a genomic library of R. jostii K01-B0171 was screened by colony hybridization, and two clones were obtained. Sequence analysis of these clones revealed that the gene cluster for lariatin biosynthesis spanning about 4.5 kb consisted of five open reading frames (larA to larE). We proposed that the linear precursor LarA is processed by LarB, LarC, and LarD, and the mature lariatin is exported by LarE.

Keywords: Lasso peptide; Lariatin; Biosynthetic pathway; Rhodococcus jostii ; Anti-tuberculosis

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production by Moo-Young Jung; Chiam Yu Ng; Hyohak Song; Jinwon Lee; Min-Kyu Oh (pp. 461-469).

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant, which was 27.4% higher than that with the parental strain. With further optimization of the medium and aeration conditions, 118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.

Keywords: 2,3-Butanediol; Enterobacter aerogenes ; Lactate dehydrogenase; Fed-batch fermentation

Elevated poly(3-hydroxybutyrate) synthesis in mutants of Ralstonia eutropha H16 defective in lipopolysaccharide biosynthesis by Ulrike Brandt; Matthias Raberg; Birgit Voigt; Michael Hecker; Alexander Steinbüchel (pp. 471-483).

Several independent transposon Tn5-induced mutants of Ralstonia eutropha H16 exhibited a poly(3-hydroxybutyric acid) (PHB) elevated phenotype and accumulated substantial amounts of PHB already in the exponential growth phase. The insertion loci of Tn5 in these six mutants were mapped in the genes hldA (twice), hldC (twice), rfaF2, and rfaF3, which are all involved in the synthesis of lipopolysaccharides (LPS), an important component of the outer membrane (OM) of Gram-negative bacteria. The generated defined deletion mutant ∆hldA confirmed the PHB elevated phenotype. According to the literature, such a truncated LPS may cause an increased permeability of the OM; thereby, the mutations may lead to a facilitated uptake of carbon source from the medium as exemplarily shown for gluconate and succinate. Thus, the ratio of carbon to nitrogen in the cell is increased. Proteome analyses revealed reinforcement of the Entner–Doudoroff pathway and of subsequent reactions that finally may lead to higher concentrations of acetyl-CoA in the cells. Due to the impaired synthesis of complete LPS, intermediates of LPS biosynthesis might be recycled by reactions yielding higher levels of NADPH and acetyl-CoA. Since the latter are precursors for synthesis of PHB, this could explain the elevated synthesis and accumulation of this polymer in case of the LPS mutants.

Keywords: Ralstonia eutropha ; Poly(3-hydroxybutyric acid); Proteome analysis; Tn5::mob mutagenesis

An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk by Takashi Soejima; Jun-ichi Minami; Tomoko Yaeshima; Keiji Iwatsuki (pp. 485-497).

Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S–23S rRNA gene, principally 2,451 bp) following EMA treatment to completely suppress the amplification of DNA of up to 7 logs (10⁷ cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25 U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1 colony forming unit (CFU)/2.22 ml within 7.5 h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1 CFU/2.22 ml).

Keywords: Viable coliform bacteria; EMA; Direct real-time PCR; Pasteurized milk

A targeted gene knockout method using a newly constructed temperature-sensitive plasmid mediated homologous recombination in Bifidobacterium longum by Kouta Sakaguchi; Jianlong He; Saori Tani; Yasunobu Kano; Tohru Suzuki (pp. 499-509).

Bifidobacteria are the main component of the human microflora. We constructed a temperature-sensitive (Ts) plasmid by random mutagenesis of the Bifidobacterium–Escherichia coli shuttle vector pKKT427 using error-prone PCR. Mutant plasmids were introduced into Bifidobacterium longum 105-A and, after screening approximately 3,000 colonies, candidate clones that grew at 30 °C but not at 42 °C were selected. According to DNA sequence analysis of the Ts plasmid, five silent and one missense mutations were found in the repB region. The site-directed mutagenesis showed only the missense mutation to be relevant to the Ts phenotype. We designated this plasmid pKO403. The Ts phenotype was also observed in B. longum NCC2705 and Bifidobacterium adolescentis ATCC15703. Single-crossover homologous-recombination experiments were carried out to determine the relationship between the length of homologous sequences encoded on the plasmid and recombination frequency: fragments greater than 1 kb gave an efficiency of more than 10³ integrations per cell. We performed gene knockout experiments using this Ts plasmid. We obtained gene knockout mutants of the pyrE region of B. longum 105-A, and determined that double-crossover homologous recombination occurred at an efficiency of 1.8 %. This knockout method also worked for the BL0033 gene in B. longum NCC2705.

Keywords: Temperature-sensitive plasmid; Bifidobacteria; Mutagenesis; Gene knockout; Homologous recombination

Isolation and characterization of a Rhodococcus strain able to degrade 2-fluorophenol by Anouk F. Duque; Syed A. Hasan; Vânia S. Bessa; Maria F. Carvalho; Ghufrana Samin; Dick B. Janssen; Paula M. L. Castro (pp. 511-520).

A pure bacterial culture able to utilize 2-fluorophenol (2-FP) as sole carbon and energy source was isolated by selective enrichment from sediments collected from a contaminated site in Northern Portugal. 16S rRNA gene analysis showed that the organism (strain FP1) belongs to the genus Rhodococcus. When grown aerobically on 2-FP, growth kinetics of strain FP1 followed the Luong model. An inhibitory effect of increasing 2-FP concentrations was observed with no growth occurring at 2-FP levels higher than ca. 4 mM. Rhodococcus strain FP1 was able to degrade a range of other organofluorine compounds, including 2-fluorobenzoate, 3-fluorobenzoate, 4-fluorobenzoate, 3-fluorophenol, 4-fluorophenol, 3-fluorocatechol, and 4-fluorocatechol, as well as chlorinated compounds such as 2-chlorophenol and 4-chlorophenol. Experiments with cell-free extracts and partially purified enzymes indicated that the first step of 2-fluorophenol metabolism was conversion to 3-fluorocatechol, suggesting an unusual pathway for fluoroaromatic metabolism. To our knowledge, this is the first time that utilization of 2-FP as a growth substrate by a pure bacterial culture is reported.

Keywords: 2-Fluorophenol (2-FP); Rhodococcus sp.; Biodegradation; Luong model; Monooxygenase

Decolorization of textile dyes by Alishewanella sp. KMK6 by Yogesh M. Kolekar; Kisan M. Kodam (pp. 521-529).

Alishewanella sp. strain KMK6 was isolated from textile dye-contaminated soil. The strain was able to decolorize and degrade different azo dyes and displayed high dye degradation ability and tolerance. The bacterium could completely degrade 2.5 g l⁻¹ dye, Reactive Blue 59 within 6 h. The induction in the level of cytochrome P-450 and activities of azoreductase and NADH–dichlorophenolindophenol reductase were observed in the cells after dye decolorization indicating the role of these enzymes. The intermediates of Reactive Blue 59 degradation were identified by high-performance liquid chromatography, gas chromatography and mass spectroscopy, and Fourier transform infrared spectroscopy. The ecotoxicity has been evaluated for dye and its metabolites by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (a yellow tetrazole) and comet assay, and it revealed that the dye degradation products were nontoxic.

Keywords: Alishewanella sp.; Reactive Blue 59; Biodegradation; Azo dyes

Insertion of transposon in the vicinity of SSK2 confers enhanced tolerance to furfural in Saccharomyces cerevisiae by Hyun-Soo Kim; Na-Rae Kim; Wankee Kim; Wonja Choi (pp. 531-540).

Furfural is one of the major inhibitors generated during sugar production from cellulosic materials and, as an aldehyde, inhibits various cellular activities of microorganisms used, leading to prolonged lag time during ethanologenic fermentation. Since Saccharomyces cerevisiae strains tolerant to furfural are of great economic benefit in producing bioethanol, much effort to obtain more efficient strains continues to be made. In this study, we examined the furfural tolerance of transposon mutant strains (Tn 1–5) with enhanced ethanol tolerance and found that one of them (Tn 2), in which SSK2 is downregulated at the transcriptional level, displayed improved furfural tolerance. Such phenotype was abolished by complementation of the entire open reading frame of SSK2, which encodes a mitogen-activated protein (MAP) kinase kinase kinase of the high osmolarity glycerol (HOG) signaling pathway, suggesting an inhibitory effect of SSK2 in coping with furfural stress. Tn 2 showed a significant decrease in the intracellular level of reactive oxygen species (ROS) and early and high activation of Hog1p, a MAP kinase integral to the HOG pathway in response to furfural. The transcriptional levels of CTT1 and GLR1, two of known Hog1p downstream target genes whose protein products are involved in reducing ROS, were increased by 43 % and 56 % respectively compared with a control strain, probably resulting in the ROS decrease. Tn 2 also showed a shortened lag time during fermentation in the presence of furfural, resulting from efficient conversion of furfural to non-toxic (or less toxic) furfuryl alcohol. Taken together, the enhanced furfural tolerance of Tn 2 is suggested to be conferred by the combined effect of an early event of less ROS accumulation and a late event of efficient detoxification of furfural.

Keywords: Furfural tolerance; SSK2 ; Furfural conversion; ROS; Hog1p activation

Bioconversion of dilute-acid pretreated sorghum bagasse to ethanol by Neurospora crassa by Ioannis Dogaris; Olga Gkounta; Diomi Mamma; Dimitris Kekos (pp. 541-550).

Bioethanol production from sweet sorghum bagasse (SB), the lignocellulosic solid residue obtained after extraction of sugars from sorghum stalks, can further improve the energy yield of the crop. The aim of the present work was to evaluate a cost-efficient bioconversion of SB to ethanol at high solids loadings (16 % at pretreatment and 8 % at fermentation), low cellulase activities (1–7 FPU/g SB) and co-fermentation of hexoses and pentoses. The fungus Neurospora crassa DSM 1129 was used, which exhibits both depolymerase and co-fermentative ability, as well as mixed cultures with Saccharomyces cerevisiae 2541. A dilute-acid pretreatment (sulfuric acid 2 g/100 g SB; 210 °C; 10 min) was implemented, with high hemicellulose decomposition and low inhibitor formation. The bioconversion efficiency of N. crassa was superior to S. cerevisiae, while their mixed cultures had negative effect on ethanol production. Supplementing the in situ produced N. crassa cellulolytic system (1.0 FPU/g SB) with commercial cellulase and β-glucosidase mixture at low activity (6.0 FPU/g SB) increased ethanol production to 27.6 g/l or 84.7 % of theoretical yield (based on SB cellulose and hemicellulose sugar content). The combined dilute-acid pretreatment and bioconversion led to maximum cellulose and hemicellulose hydrolysis 73.3 % and 89.6 %, respectively.

Keywords: Sorghum bagasse; Ethanol; Bioconversion; Cellulase; Filamentous fungi; Yeast

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Applied Microbiology and Biotechnology (v.95, #2)

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Applied Microbiology and Biotechnology (v.95, #2)