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Applied Microbiology and Biotechnology (v.82, #4)

d-Galacturonic acid catabolism in microorganisms and its biotechnological relevance by Peter Richard; Satu Hilditch (pp. 597-604).

d-Galacturonic acid is the main constituent of pectin, a naturally abundant compound. Pectin-rich residues accumulate when sugar is extracted from sugar beet or juices are produced from citrus fruits. It is a cheap raw material but currently mainly used as animal feed. Pectin has the potential to be an important raw material for biotechnological conversions to fuels or chemicals. In this paper, we review the microbial pathways for the catabolism of d-galacturonic acid that would be relevant for the microbial conversion to useful products.

Keywords: d-Galacturonic acid; d-Galacturonate; Pectin; Galactaric acid; Mucic acid; Galacturonate catabolism

Fungal laccases as tools for the synthesis of new hybrid molecules and biomaterials by Annett Mikolasch; Frieder Schauer (pp. 605-624).

Laccase is a ligninolytic enzyme widely distributed in wood-rotting fungi and which is also found in a variety of molds and insects as well as some plants and bacteria. Its biological roles range from depolmerization of lignin, coal and humic acids via the oxidation of various mono- and diaromatic structures, to polymerization reactions and pigment formation in microbial cells or spores. Apart from its action in catabolic, depolymerizing and polymerizing processes, laccases have also been shown to be powerful enzymes for coupling two different molecules to create new low-molecular-weight products in high yield. In addition to their homomolecular coupling capabilities, laccases are also able to couple a hydroxylated aromatic substrate with a nonlaccase substrate of variable structure to create new heteromolecular hybrid molecules. Thus, laccases are increasingly finding applications in biotechnology in the fields of environment-friendly synthesis of fine chemicals and for the gentle derivatization of biologically active compounds e.g., antibiotics, amino acids, antioxidants, and cytostatics. Finally, oligomerization and polymerization reactions can lead to new homo- or heteropolymers and biomaterials. These may be useful in a wide range of applications including the production of polymers with antioxidative properties, the copolymerizing of lignin components with low-molecular mass compounds, the coating of cellulosic cotton fibers or wool, the coloring of hair and leathers, or the cross-linking and oligomerization of peptides.

Keywords: Laccase; Bioactive compounds; Derivatization; Biotransformation; White biotechnology; Polymer

Metabolic effects of furaldehydes and impacts on biotechnological processes by João R. M. Almeida; Magnus Bertilsson; Marie F. Gorwa-Grauslund; Steven Gorsich; Gunnar Lidén (pp. 625-638).

There is a growing awareness that lignocellulose will be a major raw material for production of both fuel and chemicals in the coming decades—most likely through various fermentation routes. Considerable attention has been given to the problem of finding efficient means of separating the major constituents in lignocellulose (i.e., lignin, hemicellulose, and cellulose) and to efficiently hydrolyze the carbohydrate parts into sugars. In these processes, by-products will inevitably form to some extent, and these will have to be dealt with in the ensuing microbial processes. One group of compounds in this category is the furaldehydes. 2-Furaldehyde (furfural) and substituted 2-furaldehydes—most importantly 5-hydroxymethyl-2-furaldehyde—are the dominant inhibitory compounds found in lignocellulosic hydrolyzates. The furaldehydes are known to have biological effects and act as inhibitors in fermentation processes. The effects of these compounds will therefore have to be considered in the design of biotechnological processes using lignocellulose. In this short review, we take a look at known metabolic effects, as well as strategies to overcome problems in biotechnological applications caused by furaldehydes.

Keywords: Furfural; Hydroxymethylfurfural; Reductases; Bioconversion; Inhibition

Effect of various parameters on viability and growth of bacteria immobilized in sol–gel-derived silica matrices by Gisela S. Alvarez; María L. Foglia; Guillermo J. Copello; Martín F. Desimone; Luis E. Diaz (pp. 639-646).

Immobilized bacteria are being extensively used for metabolite production, biocatalysts, and biosensor construction. However, long-term viability and metabolic activity of entrapped bacteria is affected by several conditions such as their physiological state, the presence of high-osmolarity environments, porous structure and shrinkage of the matrix. The aim of this work was to evaluate the effect of various parameters on bacteria immobilized in sol–gel-derived silica matrices. With this purpose, we evaluated the stress of immobilization over bacteria cultures obtained from different growing states, the effect of cell density and bacteria capability to proliferate inside matrices. Best results to attain longer preservation times were obtained when we immobilized suspensions with an optimized bacterial number of 1 × 10⁷ cfu/gel in the presence of LB medium using aqueous silica precursors. Furthermore, the impact of osmotic stress with the subsequent intracellular trehalose accumulation and the addition of osmolites were investigated. Shorter preservation times were found for bacteria immobilized in the presence of osmolites while trehalose accumulation in stressed cells did not produce changes on entrapped bacteria viability. Finally, nutrient addition in silica matrices was studied indicating that the presence of a carbon source without the simultaneous addition of nitrogen was detrimental for immobilized E. coli. However, when both carbon and nitrogen sources were present, bacteria were able to survive longer periods of time.

Keywords: Additives; Bacteria; Escherichia coli ; Immobilization; Living cells; Sol–gel

Phenylalanine ammonia-lyase and stilbene synthase gene expression in rolB transgenic cell cultures of Vitis amurensis by K. V. Kiselev; A. S. Dubrovina; V. P. Bulgakov (pp. 647-655).

Transformation of Vitis amurensis callus culture by the plant oncogene rolB of Agrobacterium rhizogenes results in high (up to 3.15% dry wt.) levels of resveratrol in the transformed culture. The present study deals with the effect of rolB on phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in two rolB transgenic V. amurensis callus cultures with different levels of rolB expression and resveratrol production. The total expression of PALs and STSs in rolB transgenic cultures increased 1.3–3.8 times compared with the control culture. In the rolB transgenic cultures expression of VaPAL1, VaPAL2, and six STS genes was increased, while expression of VaPAL3 and VaSTS6 was not significantly changed. These results suggest that rolB increases resveratrol production via selective enhancement of expression of individual genes from PAL and STS gene families. We propose that increase of VaPAL3, VaSTS1, and VaSTS6 transcript levels is not strongly required for high resveratrol production by rolB transgenic cell cultures.

Keywords: Agrobacterium rhizogenes ; PAL ; Resveratrol; rolB gene; STS ; Vitis amurensis

Fed-batch production of unsaturated medium-chain-length polyhydroxyalkanoates with controlled composition by Pseudomonas putida KT2440 by Zhiyong Sun; Juliana A. Ramsay; Martin Guay; Bruce A. Ramsay (pp. 657-662).

Unsaturated medium-chain-length polyhydroxyalkanoates (MCL-PHA) were produced at a productivity of 0.63–1.09 g PHA l⁻¹ h⁻¹ with final PHA content ranging from 42.6 to 55.8% in single-stage, carbon-limited, fed-batch fermentations of Pseudomonas putida KT2440. A mixture of nonanoic acid (NA) and 10-undecenoic acid (UDA⁼) was fed exponentially to control growth rate. Varying the specific growth rate (0.14 h⁻¹ vs. 0.23 h⁻¹) at similar substrate feed ratios (NA:UDA⁼ = 5:1) had little effect on the final PHA content and relative composition. However, decreasing the NA:UDA⁼ ratio decreased the final amount of PHA produced from 56% with NA:UDA⁼ = 5.07:1 to only 42% at NA:UDA⁼ = 2.47:1. The molar fraction of all 3-hydroxyalkanoate monomers in the PHA product was relatively constant throughout each fermentation, indicating that the final product was homogeneous rather than a mixture of different copolymers. A linear relationship between unsaturation of the PHA produced and unsaturation of the carbon feed was found, which demonstrates the feasibility of producing unsaturated MCL-PHAs with controlled polymeric composition in a fed-batch process.

Keywords: MCL-PHA; Unsaturated; Fed-batch; Fermentation; Pseudomonas putida

Heterologous expression and characterization of a β-1,6-glucanase from Aspergillus fumigatus by A. Boisramé; C. Gaillardin (pp. 663-669).

The cell wall of Candida albicans is composed of mannoproteins associated to glycan polymers. Most of these proteins are retained in this compartment through a phosphodiester linkage between a remnant of their glycosylphosphatidylinositol anchor and the β-1,6-glucan polymer. A pure β-1,6-glucanase is thus required in order to release them. In this paper, we report the expression/secretion by the yeast Yarrowia lipolytica of an Aspergillus fumigatus enzyme homologous to previously described β-1,6-glucanases. The coding sequence was expressed under the control of a strong promoter and the recombinant enzyme was targeted to the secretory pathway using the signal sequence of a well-known major secretory protein in this host. Addition of a FLAG epitope at the C-terminus allowed its efficient purification from culture supernatant following batch adsorption. The purified enzyme was characterized as a β-1,6-glucanase and was shown to be active on C. albicans cell walls allowing the release of a previously described cell wall protein.

Keywords: β-1; 6-glucanase; Secretion; Cell wall; Fungus

Improved catalytic efficiency of Endo-β-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution by Ling Lin; Xin Meng; Pengfu Liu; Yuzhi Hong; Gaobing Wu; Xiaoluo Huang; Congcong Li; Junli Dong; Liang Xiao; Ziduo Liu (pp. 671-679).

Bacillus subtilis endo-β-1,4-glucanase (Cel5A) hydrolyzes cellulose by cleavage of the internal bonds in the glucose chains, producing new ends randomly. Using directed evolution techniques of error-prone polymerase chain reaction (PCR) and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68-fold increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78, and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme. Moreover, V74A and D272G of M44-11, which were not located in the substrate binding sites and the catalytic center, might result in improved stability and catalytic activity. These results provided useful references for directed evolution of the enzymes that belonged to the glycoside hydrolase family 5 (GH5).

Keywords: Endoglucanase; Error-prone PCR; DNA shuffling; Catalytic activity

The identification, purification, and characterization of STXF10 expressed in Streptomyces thermonitrificans NTU-88 by Hsueh-Ling Cheng; Chih-Yun Tsai; Hui-Jye Chen; Shang-Shyng Yang; Yo-Chia Chen (pp. 681-689).

Multiple xylanolytic enzymes of Streptomyces thermonitrificans NTU-88 were induced by oat-spelt xylan and separated by two-dimensional polyacrylamide and zymogram gels. Nineteen clear spots differed in pI and molecular weight values were found on the zymogram, and only spot one was seen on the corresponding silver-stained gel. These results revealed that multiple xylanases were secreted when S. thermonitrificans NTU-88 was induced and the spot (STXF10), identified as being a glycosyl hydrolase family 10 xylanase, was the predominant one among xylanases. STXF10 showed a tolerance for high temperatures and broad pH ranges and high affinity and hydrolysis efficiency for xylans. Furthermore, it also featured the minor ability to degrade different lignocellulosic substrates. Although S. thermonitrificans NTU-88 possesses multiple xylanases, our results suggest that the major form of xylanase might be selectively and specifically induced depending on the type of substrate to which the microorganism is exposed.

Keywords: Endo-β-1; 4 xylanase; Glycosyl hydrolase family 10; Streptomyces thermonitrificans NTU-88; Two-dimensional zymogram

Construction of quintuple protease gene disruptant for heterologous protein production in Aspergillus oryzae by Jaewoo Yoon; Shinya Kimura; Jun-ichi Maruyama; Katsuhiko Kitamoto (pp. 691-701).

Aspergillus oryzae has received attention as a host for heterologous protein production. However, A. oryzae has 134 protease genes, which is recognized to be one of the major reasons for the proteolytic degradation of heterologously produced proteins. We previously reported that double disruption of the protease genes (tppA and pepE) improved heterologous protein (human lysozyme) production by A. oryzae. In this study, we performed successive round of five protease genes (tppA, pepE, nptB, dppIV, and dppV) disruption in A. oryzae by pyrG marker recycling with highly efficient gene-targeting background (ΔligD). The multiple disruption of protease genes were confirmed by Southern blot analysis. Furthermore, the quintuple protease gene disruptants showed the maximum production level of bovine chymosin (CHY) that was 34% higher than those of the double protease gene disruptant (ΔtppA ΔpepE). Consequently, we successfully constructed a multiple protease gene disruptant bearing enhanced levels of CHY productivity. We presented the first evidence that the quintuple disruption of the protease genes improved the production level of a heterologous protein by A. oryzae.

Keywords: Aspergillus oryzae ; Quintuple protease gene disruption; Heterologous protein production; DNA microarray analysis; Bovine chymosin

Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant Escherichia coli by Xiao-Xing Wei; Zhen-Yu Shi; Mei-Qing Yuan; Guo-Qiang Chen (pp. 703-712).

Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (P adhE) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P adhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of P adhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.

Keywords: PHB; Polyhydroxyalkanoates; Microaerobiosis; Anaerobic promoter; phbCAB ; Alcohol dehydrogenase

Enhancement of display efficiency in yeast display system by vector engineering and gene disruption by Kouichi Kuroda; Ken Matsui; Shinsuke Higuchi; Atsushi Kotaka; Hiroshi Sahara; Yoji Hata; Mitsuyoshi Ueda (pp. 713-719).

Vector engineering and gene disruption in host cells were attempted for the enhancement of α-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2-d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology.

Keywords: Cell surface engineering; SED1 ; leu2-d ; DsRed-monomer; Display efficiency

Temperature has reciprocal effects on colanic acid and polysialic acid biosynthesis in E. coli K92 by Nicolás Navasa; Leandro Rodríguez-Aparicio; Honorina Martínez-Blanco; Mario Arcos; Miguel Ángel Ferrero (pp. 721-729).

Escherichia coli K92 is an opportunistic pathogen bacterium able to produce polysialic acid (PA) capsules when grows at 37°C. PA polysaccharides are cell-associated homopolymers tailored from acid sialic monomers that function as virulence factors in different neuroinvasive diseases caused by certain Enterobacteriaceae. Conversely, when grows at 19°C (restrictive conditions), PA synthesis was negligible, whereas in such condition, a slimy substance started to be accumulated in the culture broths. Analysis by uronic acids colorimetric determinations, gas chromatography–mass spectrometry, and Fourier transform infrared spectroscopy allowed the isolation and identification of mucoid substance as colanic acid (CA). CA is a heteropolymer containing glucose, galactose, fucose, and glucuronic acid as monomers which seems to be involved in the protection of this bacterium against environment assaults. The study of physicochemical conditions required for CA synthesis revealed that in E. coli K92, nutrient (carbon and nitrogen sources) modulates CA production, reaching the maximal values when glucose and proline were as carbon and nitrogen sources, respectively. Furthermore, we have found that E. coli K92 is able to produce CA at all temperatures tested (from 42°C to 15°C), whereas PA synthesis only occurred when bacteria were cultured at temperatures higher than 25°C. Additionally, genetic engineering approaches revealed that the CA cluster including several genes required for synthesis was placed into a DNA fragment of 100 kb using polymerase chain reaction methodology.

Keywords: Capsules; Colanic acid; Polysialic acid; Stress; Temperature regulation

A novel dichloromethane-degrading Lysinibacillus sphaericus strain wh22 and its degradative plasmid by Shijin J. Wu; Zhihang H. Hu; Lili L. Zhang; Xiang Yu; Jianmeng M. Chen (pp. 731-740).

Dichloromethane (DCM)-degrading bacterium strain wh22 (GenBank accession number FJ418643) was isolated and identified as Lysinibacillus sphaericus based on standard morphological and physiological properties, cellular fatty acid composition, mole percent guanine–cytosine content, and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. The strain also grew on many other halocarbons found in the waste gases of industrial effluents, such as 1,2-dichloroethane, chlorobromomethane, methylene bromide, 1,1,1-trichloroethane, trichloroethylene, and hexachlorobenzene. The strain harbored a novel degradative plasmid, pRC11 (48.8 kb). The genes coding for the metabolism of DCM were found to be plasmid-borne, and a physical map of the plasmid has been established. The purified plasmid was transformed to dcm ⁻ Escherichia coli DH5 at a rate of 1.65 × 10⁵. The transformed cells were able to grow on DCM at a concentration of 5–16 mM and can be further used as an excellent source for genetic manipulations leading to the construction of genetically modified microbial strains or genetically engineered microorganisms.

Keywords: Dichloromethane; Biodegradation; Degradative plasmid; L. sphaericus wh22

Development of natto with germination-defective mutants of Bacillus subtilis (natto) by Nobuo Mitsui; Hisashi Murasawa; Junichi Sekiguchi (pp. 741-748).

The effects of cortex-lysis related genes with the pdaA, sleB, and cwlD mutations of Bacillus subtilis (natto) NAFM5 on sporulation and germination were investigated. Single or double mutations did not prevent normal sporulation, but did affect germination. Germination was severely inhibited by the double mutation of sleB and cwlD. The quality of natto made with the sleB cwlD double mutant was tested, and the amounts of glutamic acid and ammonia were very similar to those in the wild type. The possibility of industrial development of natto containing a reduced number of viable spores is presented.

Keywords: Germination; sleB ; cwlD ; pdaA ; Bacillus subtilis ; Natto

A fluorescent, genetically engineered microorganism that degrades organophosphates and commits suicide when required by Qin Li; Yi-Jun Wu (pp. 749-756).

One way to reduce the potential risk of genetically engineered microorganisms (GEMs) to the environment is to use a containment system that does not interfere with the performance of the GEM until activated. Such a system can be created by inserting a suicide cassette consisting of a toxin-encoding gene controlled by an inducible promoter. We constructed a GEM that can degrade organophosphorus compounds, emit green fluorescence, and commit suicide when required by putting the genes that control these different functions under different promoters. The genes for enhanced green fluorescent protein (EGFP) and organophosphorus hydrolase (OPH) were cloned downstream of the lambda P_L promoter in the plasmid pBV220. These genes could be expressed freely as long as the GEM was metabolizing because the repressor sequence cIts857 had been deleted. The extracellular nuclease gene of Serratia marcescens, without its leader-coding sequence, provided the suicide mechanism. This was put under the control of the T7 promoter to form a suicide cassette activated by the presence of an environmental signal, in this case, arabinose. To improve the reliability of this containment system, the suicide cassette was duplicated within the conditional suicide plasmid. The plasmid carrying the EGFP and OPH fusion genes and that containing the suicide cassette were compatible and coexisted in the same host.

Keywords: Genetically engineered bacteria; Suicide; Organophosphorus hydrolase; Green fluorescent protein

Dry anaerobic ammonia–methane production from chicken manure by Fatma Abouelenien; Yoshiaki Kitamura; Naomichi Nishio; Yutaka Nakashimada (pp. 757-764).

The effect of temperature on production of ammonia during dry anaerobic fermentation of chicken manure (CM), inoculated with thermophilic methanogenic sludge, was investigated in a batch condition for 8 days. Incubation temperature did not have a significant effect on the production of ammonia. Almost complete inhibition of production of methane occurred at 55 and 65°C while quite low yields of 8.45 and 6.34 ml g⁻¹ VS (volatile solids) were observed at 35 and 45°C due to a higher accumulation of ammonia. In order to improve the production of methane during dry anaerobic digestion of CM, stripping of ammonia was performed firstly on the CM previously fermented at 65°C for 8 days: the stripping for 1 day at 85°C and pH 10 removed 85.5% of ammonia. The first-batch fermentation of methane for 75 days was conducted next, using the ammonia-stripped CM inoculated with methanogenic sludge at different ratios, (CM: thermophilic sludge) of 1:2, 1:1, and 2:1 on volume per volume basis at both 35 and 55°C. Production of methane improved and was higher than that of the control (without stripping of ammonia) but the yield of 20.4 ml g⁻¹ VS was still low, so second stripping of ammonia was conducted, which resulted in 74.7% removal of ammonia. A great improvement in the production of methane of 103.5 ml g⁻¹ VS was achieved during the second batch for 55 days.

Keywords: Ammonia production; Ammonia stripping; Chicken manure; Dry fermentation; Methane fermentation

Construction of an Escherichia coli to Bacillus thuringiensis shuttle vector for large DNA fragments by Xiaoyan Liu; Donghai Peng; Yi Luo; Lifang Ruan; Ziniu Yu; Ming Sun (pp. 765-772).

Shuttle vectors for Bacillus thuringiensis or Bacillus cereus usually cannot hold fragments larger than 20 kb. With the development of genome research, shuttle vectors with higher loading capacity are necessary. We constructed an Escherichia coli to B. thuringiensis shuttle vector, pEMB0557, with a large loading capacity. This vector incorporated the ori60 replicon from B. thuringiensis subsp. kurstaki YBT-1520, erythromycin resistance (B. thuringiensis), and chloromycetin resistance (E. coli) genes. A bacterial artificial chromosome library of B. thuringiensis strain CT-43 was constructed and pEMB0557 was able to accommodate at least a 70-kb DNA fragment. Simultaneously, the cry1B gene on a 40-kb fragment could express a 140-kDa protein in plasmid-cured B. thuringiensis BMB171. Due to its high capacity and utility in expressing exogenous genes, pEMB0557 will be useful in cloning (especially silencing genes) and expressing large DNA fragments (e.g., gene clusters) in B. thuringiensis. Plasmid pEMB0557 provides a new tool for B. thuringiensis genome or B. cereus group research.

Keywords: Bacillus thuringiensis ; Shuttle vector; Large holding capability; Genome research

Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm by Alberto Mariscal; Rosa M. Lopez-Gigosos; Manuel Carnero-Varo; Joaquin Fernandez-Crehuet (pp. 773-783).

A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.

Keywords: Biofilm; Resazurin; Disinfectant; Fluorescence

Optimization of preservation conditions of As (III) bioreporter bacteria by Anke Kuppardt; Antonis Chatzinotas; Uta Breuer; Jan Roelof van der Meer; Hauke Harms (pp. 785-792).

Long-term preservation of bioreporter bacteria is essential for the functioning of cell-based detection devices, particularly when field application, e.g., in developing countries, is intended. We varied the culture conditions (i.e., the NaCl content of the medium), storage protection media, and preservation methods (vacuum drying vs. encapsulation gels remaining hydrated) in order to achieve optimal preservation of the activity of As (III) bioreporter bacteria during up to 12 weeks of storage at 4°C. The presence of 2% sodium chloride during the cultivation improved the response intensity of some bioreporters upon reconstitution, particularly of those that had been dried and stored in the presence of sucrose or trehalose and 10% gelatin. The most satisfying, stable response to arsenite after 12 weeks storage was obtained with cells that had been dried in the presence of 34% trehalose and 1.5% polyvinylpyrrolidone. Amendments of peptone, meat extract, sodium ascorbate, and sodium glutamate preserved the bioreporter activity only for the first 2 weeks, but not during long-term storage. Only short-term stability was also achieved when bioreporter bacteria were encapsulated in gels remaining hydrated during storage.

Keywords: Bioreceptor; Arsenic; Preservation; Storage

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Applied Microbiology and Biotechnology (v.82, #4)

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Applied Microbiology and Biotechnology (v.82, #4)