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Applied Microbiology and Biotechnology (v.82, #2)

Diatoms in biotechnology: modern tools and applications by Andrew Bozarth; Uwe-G. Maier; Stefan Zauner (pp. 195-201).

Diatoms have played a decisive role in the ecosystem for millions of years as one of the foremost set of oxygen synthesizers on earth and as one of the most important sources of biomass in oceans. Previously, diatoms have been almost exclusively limited to academic research with little consideration of their practical uses beyond the most rudimentary of applications. Efforts have been made to establish them as decisively useful in such commercial and industrial applications as the carbon neutral synthesis of fuels, pharmaceuticals, health foods, biomolecules, materials relevant to nanotechnology, and bioremediators of contaminated water. Progress in the technologies of diatom molecular biology such as genome projects from model organisms, as well as culturing conditions and photobioreactor efficiency, may be able to be combined in the near future to make diatoms a lucrative source of novel substances with widespread relevance.

Keywords: Diatom; Nanotechnology; Biofuel; Bioremediation

Versatile capacity of shuffled cytochrome P450s for dye production by Nedeljka N. Rosic (pp. 203-210).

DNA family shuffling is a relatively new method of directed evolution used to create novel enzymes in order to improve their existing properties or to develop new features. This method of evolution in vitro has one basic requirement: a high similarity of initial parental sequences. Cytochrome P450 enzymes are relatively well conserved in their amino acid sequences. Members of the same family can have more than 40% of sequence identity at the protein level and are therefore good candidates for DNA family shuffling. These xenobiotic-metabolising enzymes have an ability to metabolise a wide range of chemicals and produce a variety of products including blue pigments such as indigo. By applying the specifically designed DNA family shuffling approach, catalytic properties of cytochrome P450 enzymes were further extended in the chimeric progeny to include a new range of blue colour formations. This mini-review evokes the possibility of exploiting directed evolution of cytochrome P450s and the novel enzymes created by DNA family shuffling for the production of new dyes.

Keywords: Directed evolution; DNA family shuffling; CYP; Indole metabolism; Molecular breeding; Indigo

Inulinase-expressing microorganisms and applications of inulinases by Zhenming Chi; Zhe Chi; Tong Zhang; Guanglei Liu; Lixi Yue (pp. 211-220).

In this review article, inulinase-expressing microorganisms and its potential applications in transformation of inulin into very-high-fructose syrup, bioethanol, and inulooligosaccharides are overviewed. In the past 10 years, many new inulinase producers have been obtained and many genes encoding inulinases from different microorganisms have been cloned and characterized. Some novel processes for exoinulinase overproduction have been developed for bioethanol production and ultra-high-fructose syrup. The endoinulinases have also been used for production of inulooligosaccharides from inulin and inulin-containing materials.

Keywords: Inulin; Inulinase genes; Bioethanol; Fructose; Inulooligosaccharides

Advanced control of dissolved oxygen concentration in fed batch cultures during recombinant protein production by A. Kuprijanov; S. Gnoth; R. Simutis; A. Lübbert (pp. 221-229).

Design and experimental validation of advanced pO₂ controllers for fermentation processes operated in the fed-batch mode are described. In most situations, the presented controllers are able to keep the pO₂ in fermentations for recombinant protein productions exactly on the desired value. The controllers are based on the gain-scheduling approach to parameter-adaptive proportional-integral controllers. In order to cope with the most often appearing distortions, the basic gain-scheduling feedback controller was complemented with a feedforward control component. This feedforward/feedback controller significantly improved pO₂ control. By means of numerical simulations, the controller behavior was tested and its parameters were determined. Validation runs were performed with three Escherichia coli strains producing different recombinant proteins. It is finally shown that the new controller leads to significant improvements in the signal-to-noise ratio of other key process variables and, thus, to a higher process quality.

Keywords: Fermentation; Dissolved oxygen; Adaptive pO₂ control; Gain scheduling approach

Xylose anaerobic conversion by open-mixed cultures by Margarida F. Temudo; Tania Mato; Robbert Kleerebezem; Mark C. M. van Loosdrecht (pp. 231-239).

Xylose is, after glucose, the dominant sugar in agricultural wastes. In anaerobic environments, carbohydrates are converted into volatile fatty acids and alcohols. These can be used as building blocks in biotechnological or chemical processes, e.g., to produce bioplastics. In this study, xylose fermentation by mixed microbial cultures was investigated and compared with glucose under the same conditions. The product spectrum obtained with both substrates was comparable. It was observed that, in the case of xylose, a higher fraction of the carbon was converted into catabolic products (butyrate, acetate, and ethanol) and the biomass yield was approximately 20% lower than on glucose, 0.16 versus 0.21 Cmol X/Cmol S. This lower yield is likely related to the need of an extra ATP during xylose uptake. When submitted to a pulse of glucose, the population cultivated on xylose could instantaneously convert the glucose. No substrate preference was observed when glucose and xylose were fed simultaneously to the continuously operated bioreactor.

Keywords: Xylose; Glucose; Fermentation; Bulk chemicals; Hydrogen; Mixed microbial population

Long-term production of soluble human Fas ligand through immobilization of Dictyostelium discoideum in a fibrous bed bioreactor by Jie Chen; Heming Chen; Xiangchen Zhu; Yinghua Lu; Shang-Tian Yang; Zhinan Xu; Peilin Cen (pp. 241-248).

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.

Keywords: Dictyostelium discoideum ; Immobilization; Soluble human Fas ligand; Fibrous bed bioreactor

Impact of oxygen supply on rtPA expression in Escherichia coli BL21 (DE3): ammonia effects by Hengwei Wang; Fengqing Wang; Dongzhi Wei (pp. 249-259).

In shake flasks, good oxygen supply tended to decrease rtPA expression in media containing only yeast extract and tryptone, while oxygen limitation would increase rtPA synthesis in the same medium. Our data showed that though the drop of rtPA expression in the 20-ml cultures of LBG or 2YTG was accompanied with a severe acetate accumulation, it was actually caused by low ammonia. The rtPA expression level could be significantly improved by increasing culture ammonium ion up to 500 mM. The effects of exogenous high ammonia on cell growth and rtPA expression were further examined in shake flasks and a 4-l fermentor. The high ammonia had no significant impact on cell growth and oxygen respiratory activity but significantly depressed the activities of glutamine synthetase/glutamate synthase and glutamate dehydrogenase, suggesting that ammonium ion as a nitrogen source improved the protein expression by mediating ammonia-assimilating enzymes. We thus propose in our work that E. coli cells, which were grown to a certain density to produce rtPA, would undergo nitrogen starvation under the low ammonia conditions even when the organic nitrogen sources remained abundant. The scale-up of rtPA production from shake flasks to fermentors could be readily achieved in the media containing rich ammonium ion.

Keywords: Escherichia coli ; Ammonia; Scale-up; Fed-batch cultivation; Recombinant protein expression; rtPA

Molecular and biochemical characterization of a distinct tyrosinase involved in melanin production from Aeromonas media by Xia Wan; Baozhong Chai; Yi Liao; Ying Su; Tao Ye; Ping Shen; Xiangdong Chen (pp. 261-269).

A new tyrosinase was isolated from Aeromonas media strain WS and purified to homogeneity. The purified tyrosinase, termed TyrA, had a molecular mass of 58 kDa and an isoelectric point of 4.90. It exhibited optimal monophenol and diphenol oxidase activities under basic conditions (pH > 8.0). TyrA had a relatively higher affinity to diphenol substrate l-dihydroxyphenylalanine (l-dopa) than many other tyrosinases. EDTA or glutathione notably inhibited the enzymatic activities of TyrA, whereas Triton X-100 and SDS activated them. The full-length TyrA gene was cloned, and it encodes a 518 amino acid protein with little similarities to other reported tyrosinases. However, the purified recombinant TyrA expressed in Escherichia coli demonstrated tyrosinase activity. These results suggest that TyrA is the first reported distinct tyrosinase involved in melanin production in the genus Aeromonas.

Keywords: Aeromonas media ; Tyrosinase; Melanin; Diphenol oxidase; Monophenol oxidase

The cyanide hydratase from Neurospora crassa forms a helix which has a dimeric repeat by Kyle C. Dent; Brandon W. Weber; Michael J. Benedik; B. Trevor Sewell (pp. 271-278).

The fungal cyanide hydratases form a functionally specialized subset of the nitrilases which catalyze the hydrolysis of cyanide to formamide with high specificity. These hold great promise for the bioremediation of cyanide wastes. The low resolution (3.0 nm) three-dimensional reconstruction of negatively stained recombinant cyanide hydratase fibers from the saprophytic fungus Neurospora crassa by iterative helical real space reconstruction reveals that enzyme fibers display left-handed D₁ S_5.4 symmetry with a helical rise of 1.36 nm. This arrangement differs from previously characterized microbial nitrilases which demonstrate a structure built along similar principles but with a reduced helical twist. The cyanide hydratase assembly is stabilized by two dyadic interactions between dimers across the one-start helical groove. Docking of a homology-derived atomic model into the experimentally determined negative stain envelope suggests the location of charged residues which may form salt bridges and stabilize the helix.

Keywords: Cyanide; Nitrilase; 3d protein reconstruction; Cyanide hydratase

Improving the thermostability of N-carbamyl-d-amino acid amidohydrolase by error-prone PCR by Hong Yu; Jian Li; Dalong Zhang; Yunliu Yang; Weihong Jiang; Sheng Yang (pp. 279-285).

To facilitate the easier production of d-amino acids using N-carbamyl-d-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 °C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of d-amino acids.

Keywords: N-carbamyl-d-amino acid amidohydrolase; Thermostability; d-amino acid; Error-prone PCR

Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production by Zhong-peng Guo; Liang Zhang; Zhong-yang Ding; Zheng-Xiang Wang; Gui-Yang Shi (pp. 287-292).

The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD⁺-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1Δ and gpd2Δ were studied under alcoholic fermentation conditions. gpd1Δ mutants exhibited a 4.29% (relative to the amount of substrate consumed) decrease in glycerol production and 6.83% (relative to the amount of substrate consumed) increased ethanol yield while gpd2Δ mutants exhibited a 7.95% (relative to the amount of substrate consumed) decrease in glycerol production and 7.41% (relative to the amount of substrate consumed) increased ethanol yield compared with the parental strain. The growth rate of the two mutants were slightly lower than that of the wild type under the exponential phase whereas ANG1 (gpd1Δ) and the decrease in glycerol production was not accompanied by any decline in the protein content of the strain ANG1 (gpd1Δ) but a slight decrease in the strain ANG2 (gpd2Δ). Meanwhile, dramatic decrease of acetate acid formation was observed in strain ANG1 (gpd1Δ) and ANG2 (gpd2Δ) compared to the parental strain. Therefore, it is possible to improve the ethanol yield by interruption of glycerol pathway in industrial alcoholic yeast.

Keywords: Saccharomyces cerevisiae ; Glycerol-3-phosphate dehydrogenase; Glycerol production; Ethanol yield

Cell-surface modification of non-GMO without chemical treatment by novel GMO-coupled and -separated cocultivation method by Natsuko Miura; Wataru Aoki; Naoki Tokumoto; Kouichi Kuroda; Mitsuyoshi Ueda (pp. 293-301).

We developed a novel method to coat living non-genetically modified (GM) cells with functional recombinant proteins. First, we prepared GM yeast to secrete constructed proteins that have two domains: a functional domain and a binding domain that recognizes other cells. Second, we cocultivated GM and non-GM yeasts that share and coutilize the medium containing recombinant proteins produced by GM yeasts using a filter-membrane-separated cultivation reactor. We confirmed that GM yeast secreted enhanced green fluorescent protein (EGFP) fusion proteins to culture medium. After cocultivation, EGFP fusion proteins produced by GM yeast were targeted to non-GM yeast (Saccharomyces cerevisiae BY4741ΔCYC8 strain) cell surface. Yeast cell-surface engineering is a useful method that enables the coating of GM yeast cell surface with recombinant proteins to produce highly stable and accumulated protein particles. The results of this study suggest that development of cell-surface engineering from GM organisms (GMOs) to living non-GMOs by our novel cocultivation method is possible.

Keywords: Non-GMO (genetically modified organism); Cocultivation; Cell-surface engineering; MSSM (molecular sniping and shooting method)

Analysis of functions in plasmid pHZ1358 influencing its genetic and structural stability in Streptomyces lividans 1326 by Yuhui Sun; Xinyi He; Jingdan Liang; Xiufen Zhou; Zixin Deng (pp. 303-310).

The complete DNA sequence of plasmid pHZ1358, a widely used vector for targeted gene disruption and replacement experiments in many Streptomyces hosts, has been determined. This has allowed a detailed analysis of the basis of its structural and segregational instability, compared to the high copy number plasmid pIJ101 of Streptomyces lividans 1326 from which it was derived. A 574-bp DNA region containing sti (strong incompatibility locus) was found to be a determinant for segregational instability in its original S. lividans 1326 host, while the structural instability was found to be related to the facile deletion of the entire Escherichia coli-derived part of pHZ1358, mediated by recombination between 36-bp direct repeats. A point mutation removing the BamHI site inside the rep gene encoding a replication protein (rep*) and/or a spontaneous deletion of the 694-bp region located between rep and sti including the uncharacterized ORF85 (orf85 ⁻) produced little or no effect on stability. A pHZ1358 derivative (pJTU412, sti −, rep*, orf85 ⁻) was then constructed which additionally lacked one of the 36-bp direct repeats. pJTU412 was demonstrated to be structurally stable but segregationally unstable and, in contrast to sti ⁺ pHZ1358, allowed efficient targeted gene replacement in S. lividans 1326.

Keywords: pIJ101; pHZ1358; Streptomyces lividans ; Gene replacements

Cell wall adaptations of planktonic and biofilm Rhodococcus erythropolis cells to growth on C5 to C16 n-alkane hydrocarbons by Carla C. C. R. de Carvalho; Lukas Y. Wick; Hermann J. Heipieper (pp. 311-320).

Rhodococcus erythropolis was found to utilize C5 to C16 n-alkane hydrocarbons as sole source of carbon and energy when growing as planktonic or biofilm cells attached to polystyrene surfaces. Growth rates on even numbered were two- to threefold increased relatively to odd-numbered n-alkanes and depended on the chain length of the n-alkanes. C10-, C12-, C14-, and C16-n-alkanes gave rise to two- to fourfold increased maximal growth rates relative to C5- to C9-hydrocarbons. In reaction to the extremely poor water solubility of the n-alkanes, both planktonic and biofilm cells exhibited distinct adaptive changes. These included the production of surface active compounds and substrate-specific modifications of the physicochemical cell surface properties as expressed by the microbial adhesion to hydrocarbon- and contact angle-based hydrophobicity, as well as the zeta potential of the cells. By contrast, n-alkane-specific alterations of the cellular membrane composition were less distinct. The specificity of the observed autecological changes suggest that R. erythropolis cells may be used in the development and application of sound biocatalytic processes using n-alkanes as substrates or substrate reservoirs or for target-specific bioremediation of oils and combustibles, respectively.

Keywords: Cell adhesion; Surface properties; Fatty acids; Surface tension; Cell charge

Short chain regioselectively hydrolyzed scleroglucans induce maturation of porcine dendritic cells by Diane Bimczok; Julika Wrenger; Thomas Schirrmann; Hermann-Josef Rothkötter; Victor Wray; Udo Rau (pp. 321-331).

Branched β-1,3/1,6-glucans (scleroglucan) were produced by cultivation of Sclerotium rolfsii ATCC 15205. Regioselective hydrolysis at the β-1,3-linkage of the cell-free and purified polysaccharide was performed in borosilicate glass bottles at pH 5, 121°C, and 1 bar for 72 h. The mixture was divided into four molar mass fractions by stepwise cross-flow filtration using different cutoffs. In vitro studies revealed that scleroglucan hydrolysates with a low molar mass of less than 5 kDa significantly stimulated the activation and maturation of porcine monocyte derived dendritic cells (MoDC) by upregulation of CD40 and CD80/86 as well as by reduction of antigen uptake. MoDC treated with low molar mass scleroglucan showed a considerable increase in the amounts of secreted proinflammatory cytokine tumor necrosis factor alpha and stimulated the proliferation of lymphocytes. Therefore, scleroglucan molecules of low molecular weight are able to induce activation and maturation of porcine DC, which are key initiators of inflammatory and adaptive immune responses, and could provide improved protection against infectious diseases.

Keywords: Porcine dendritic cells; Cytokines; β-Glucan; Scleroglucan; Regioselective hydrolysis

Use of allylthiourea to produce soluble methane monooxygenase in the presence of copper by Yinghao Yu; Juliana A. Ramsay; Bruce A. Ramsay (pp. 333-339).

Methanotrophs expressing soluble methane monooxygenase (sMMO) may find use in a variety of industrial applications. However, sMMO expression is strongly inhibited by copper, and the growth rate may be limited by the aqueous solubility of methane. In this study, addition of allylthiourea decreased intracellular copper in Methylosinus trichosporium OB3b, allowing sMMO production at Cu/biomass ratios normally not permitting sMMO synthesis. The presence of about 1.5 μmoles intracellular Cu g⁻¹ dry biomass resulted in sMMO activity of about 250 μmoles 1-napthol formed per hour gram dry biomass whether this intracellular Cu concentration was achieved by Cu limitation or by allylthiourea addition. No loss of sMMO activity occurred when the growth substrate was switched from methane to methanol when allylthiourea had been added to growth medium containing copper. Addition of copper to medium that was almost copper-free increased the yield of dry biomass from methanol from 0.20 to 0.36 g g⁻¹, demonstrating that some copper was necessary for good growth. This study demonstrated a method by which sMMO can be produced by M. trichosporium OB3b while growing on methanol in copper-containing medium.

Keywords: Allylthiourea; Methylosinus trichosporium OB3b; sMMO; Copper; Methane; Methanol

Enhanced transformation of malachite green by laccase of Ganoderma lucidum in the presence of natural phenolic compounds by Kumarasamy Murugesan; In-Hee Yang; Young-Mo Kim; Jong-Rok Jeon; Yoon-Seok Chang (pp. 341-350).

In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l⁻¹) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly (p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization of MG.

Keywords: Ganoderma lucidum ; Laccase; Malachite green; Decolorization; Natural phenolic compounds

Edible oil degradation by using yeast coculture of Rhodotorula pacifica ST3411 and Cryptococcus laurentii ST3412 by Daisuke Sugimori (pp. 351-357).

To develop a microbial treatment of edible oil-contaminated wastewater, microorganisms capable of rapidly degrading edible oil were screened. The screening study yielded a yeast coculture comprising Rhodotorula pacifica strain ST3411 and Cryptococcus laurentii strain ST3412. The coculture was able to degrade efficiently even at low contents of nitrogen ([NH₄–N] = 240 mg/L) and phosphorus sources ([PO₄–P] = 90 mg/L). The 24-h degradation rate of 3,000 ppm mixed oils (salad oil/lard/beef tallow, 1:1 w/w) at 20°C was 39.8% ± 9.9% (means ± standard deviations of eight replicates). The highest degradation rate was observed at 20°C and pH 8. In a scaled-up experiment, the salad oil was rapidly degraded by the coculture from 671 ± 52.0 to 143 ± 96.7 ppm in 24 h, and the degradation rate was 79.4% ± 13.8% (means ± standard deviations of three replicates). In addition, a repetitive degradation was observed with the cell growth by only pH adjustment without addition of the cells.

Keywords: Oil degradation; Coculture; Rhodotorula; Cryptococcus; Wastewater treatment

Indigenous microfungi and plants reduce soil nonylphenol contamination and stimulate resident microfungal communities by Mariangela Girlanda; Sergio Enrico Favero-Longo; Alexandra Lazzari; Rossana Segreto; Silvia Perotto; Consolata Siniscalco (pp. 359-370).

Nonylphenol, the most abundant environmental pollutant with endocrine disrupting activity, is also toxic to plants and microorganisms, but its actual impact in the field is unknown. In this study, diversity of culturable soil microfungal and plant communities was assessed in a disused industrial estate, at three sites featuring different nonylphenol pollution. Although soil microfungal assemblages varied widely among the sites, no significant correlation was found with point pollutant concentrations, thus suggesting indirect effects of soil contamination on microfungal assemblages. The potential of indigenous fungi and plants to remove nonylphenol was assessed in mesocosm experiments. Poplar plants and a fungal consortium consisting of the most abundant strains in the nonylphenol-polluted soil samples were tested alone or in combination for their ability to reduce, under greenhouse conditions, nonylphenol levels either in a sterile, artificially contaminated sand substrate, or in two non-sterile soils from the original industrial area. Introduction of indigenous fungi consistently reduced nonylphenol levels in all substrates, up to ca. 70% depletion, whereas introduction of the plant proved to be effective only with high initial pollutant levels. In native non-sterile soil, nonylphenol depletion following fungal inoculation correlated with biostimulation of indigenous fungi, suggesting positive interactions between introduced and resident fungi.

Keywords: Rhizoremediation; Bioaugmentation; Soil microfungi; Endocrine disrupters

New protocol for the rapid quantification of exopolysaccharides in continuous culture systems of acidophilic bioleaching bacteria by Caroline Michel; Claire Bény; Fabian Delorme; Laurence Poirier; Pauline Spolaore; Dominique Morin; Patrick d’Hugues (pp. 371-378).

In this study, we investigate exopolysaccharide production by a bacterial consortium during the bioleaching of a cobaltiferrous pyrite. Whereas comparable studies have looked at exopolysaccharide production in batch systems, this study focuses on a continuous system comprising a series of four stirred bioreactors and reveals the difficulties in quantifying biomolecules in complex media such as bioleached samples. We also adapted the phenol/sulphuric acid method to take into account iron interference, thus establishing a new protocol for sugar quantification in bioleached samples characterised by low pH (1.4) and high iron concentration (2 g l⁻¹). This allows sugar analysis without any prior sample preparation step; only a small amount of sample is needed (0.5 ml) and sample preparation is limited to a single filtration step. We found that free exopolysaccharides represented more than 80% of the total sugars in the bioreactors, probably because stirring creates abrasive conditions and detaches sugars bound to pyrite or bacteria and that they were produced mainly in the first two reactors where bioleaching activity was greatest. However, we could not establish any direct link between the measured exopolysaccharide concentration and bioleaching activity. Exopolysaccharides could have another role (protection against stress) in addition to that in bacterial attachment.

Keywords: Exopolysaccharides; Bioleaching; Laboratory-scale continuous bioreactors; Acidophilic bacterial consortium; Modified phenol/sulphuric acid method; ATR-FTIR

Optimization of actinomycin V production by Streptomyces triostinicus using artificial neural network and genetic algorithm by Vineeta Singh; Mahvish Khan; Saif Khan; C. K. M. Tripathi (pp. 379-385).

Artificial neural network (ANN) and genetic algorithm (GA) were applied to optimize the medium components for the production of actinomycinV from a newly isolated strain of Streptomyces triostinicus which is not reported to produce this class of antibiotics. Experiments were conducted using the central composite design (CCD), and the data generated was used to build an artificial neural network model. The concentrations of five medium components (MgSO₄, NaCl, glucose, soybean meal and CaCO₃) served as inputs to the neural network model, and the antibiotic yield served as outputs of the model. Using the genetic algorithm, the input space of the neural network model was optimized to find out the optimum values for maximum antibiotic yield. Maximum antibiotic yield of 452.0 mg l⁻¹ was obtained at the GA-optimized concentrations of medium components (MgSO₄ 3.657; NaCl 1.9012; glucose 8.836; soybean meal 20.1976 and CaCO₃ 13.0842 gl⁻¹). The antibiotic yield obtained by the ANN/GA was 36.7% higher than the yield obtained with the response surface methodology (RSM).

Keywords: Actinomycin V; Streptomyces ; Central composite design; Neural network; Genetic algorithm

Efficient generation of recessive traits in diploid sake yeast by targeted gene disruption and loss of heterozygosity by Atsushi Kotaka; Hiroshi Sahara; Akihiko Kondo; Mitsuyoshi Ueda; Yoji Hata (pp. 387-395).

Sake yeast, a diploid Saccharomyces cerevisiae strain, is useful for industry but difficult to genetically engineer because it hardly sporulates. Until now, only a few recessive mutants of sake yeast have been obtained. To solve this problem, we developed the high-efficiency loss of heterozygosity (HELOH) method, which applies a two-step gene disruption. First, a heterozygous disruptant was constructed by gene replacement with URA3, followed by marker recycling on medium containing 5-fluoroorotic acid (5-FOA). Subsequently, spontaneous loss of heterozygosity (LOH) yielding a homozygous disruptant was selected for in a second round of gene integration. During this step, the wild-type allele of the heterozygous disruptant was marked by URA3 integration, and the resulting transformants were cultivated in non-selective medium to induce recombination and then grown on medium with 5-FOA to enrich for mutants that had undergone LOH. Although the frequency with which LOH occurs is extremely low, many homozygous disruptants were obtained with the HELOH method. Thus, we were able to efficiently construct homozygous disruptants of diploid sake yeast without sporulation, and sake yeast strains with multiple auxotrophies and a protease deficiency could be constructed. The HELOH method, therefore, facilitated the utilization of diploid sake yeast for genetic engineering purposes.

Keywords: Homozygous disruptant; Diploid sake yeast; Marker recycling; Loss of heterozygosity

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Applied Microbiology and Biotechnology (v.82, #2)

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Applied Microbiology and Biotechnology (v.82, #2)