Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.56, #3-4)

Biotechnological production of itaconic acid by Th. Willke; K.-D. Vorlop (pp. 289-295).

Itaconic acid (IA) is an unsaturated dicarbonic organic acid. It can easily be incorporated into polymers and may serve as a substitute for petrochemical-based acrylic or methacrylic acid. It is used at 1–5% as a comonomer in resins and also in the manufacture of synthetic fibres, in coatings, adhesives, thickeners and binders. The favoured production process is fermentation of carbohydrates by fungi, with a current market volume of about 15,000 t/a. Due to the high price of about US$4/kg, the use of IA is restricted. At present, the production rates do not exceed 1 g l^–1 h^–1, accompanied by product concentrations of about 80 g l^–1. New biotechnology approaches, such as immobilisation techniques, screening programmes and genetic engineering, could lead to higher productivity. Also, the use of alternative substrates may reduce costs and thus open the market for new and increased applications.

Biotechnological production of vanillin by H. Priefert; J. Rabenhorst; A. Steinbüchel (pp. 296-314).

Vanillin is one of the most important aromatic flavor compounds used in foods, beverages, perfumes, and pharmaceuticals and is produced on a scale of more than 10 thousand tons per year by the industry through chemical synthesis. Alternative biotechnology-based approaches for the production are based on bioconversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino acids, and on de novo biosynthesis, applying fungi, bacteria, plant cells, or genetically engineered microorganisms. Here, the different biosynthesis routes involved in biotechnological vanillin production are discussed.

Bacterial alginate: physiology, product quality and process aspects by W. Sabra; A.-P. Zeng; W.-D. Deckwer (pp. 315-325).

Alginate, a copolymer of β-D-mannuronic acid and α-L-guluronic acid and currently commercially produced from the marine brown algae, can also be biologically produced by bacteria such as Azotobacter vinelandii, A. chroococcum and several species of Pseudomonas. The ever-increasing applications of this polymer in the food and pharmaceutical sectors have led to continuing research interest aimed at better understanding the metabolic pathways, the physiological or biological function of this polymer, the regulation of its formation and composition, and optimising the microbial production process. These aspects are reviewed here, with particular attention to alginate formation in the soil bacterium A. vinelandii. In addition, the biotechnological and industrial applications of alginate are summarised.

Microbial xylanases and their industrial applications: a review by Q. Beg; M. Kapoor; L. Mahajan; G. Hoondal (pp. 326-338).

Despite an increased knowledge of microbial xylanolytic systems in the past few years, further studies are required to achieve a complete understanding of the mechanism of xylan degradation by microorganisms and their enzymes. The enzyme system used by microbes for the metabolism of xylan is the most important tool for investigating the use of the second most abundant polysaccharide (xylan) in nature. Recent studies on microbial xylanolytic systems have generally focussed on induction of enzyme production under different conditions, purification, characterization, molecular cloning and expression, and use of enzyme predominantly for pulp bleaching. Rationale approaches to achieve these goals require a detailed knowledge of the regulatory mechanism governing enzyme production. This review will focus on complex xylan structure and the microbial enzyme complex involved in its complete breakdown, studies on xylanase regulation and production and their potential industrial applications, with special reference to biobleaching.

Microbial degradation and fate in the environment of methyl tert-butyl ether and related fuel oxygenates by F. Fayolle; J.-P. Vandecasteele; F. Monot (pp. 339-349).

Oxygenates, mainly methyl tert-butyl ether (MTBE), are commonly added to gasoline to enhance octane index and improve combustion efficiency. Other oxygenates used as gasoline additives are ethers such as ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME), and alcohols such as tert-butyl alcohol (TBA). As a result of its wide use, MTBE has been detected, mainly in the USA, in groundwater and surface waters, and is a cause of concern because of its possible health effects and other undesirable consequences. MTBE is a water-soluble and mobile compound that generates long pollution plumes in aquifers impacted by gasoline releases from leaking tanks. Field observations concur in estimating that, because of recalcitrance to biodegradation, natural attenuation is slow (half-life of at least 2 years). However, quite significant advances have been made in recent years concerning the microbiology of the degradation of MTBE and other oxygenated gasoline additives. The recalcitrance of these compounds results from the presence in their structure of an ether bond and of a tertiary carbon structure. For the most part, only aerobic microbial degradation systems have been reported so far. Consortia capable of mineralizing MTBE have been selected. Multiple instances of the cometabolism of MTBE with pure strains or with microfloræ, growing on n-alkanes, isoalkanes, cyclohexane or ethers (diethyl ether, ETBE), have been described. MTBE was converted into TBA in all cases and was sometimes further degraded, but it was not used as a carbon source by the pure strains. However, mineralization of MTBE and TBA by several pure bacterial strains using these compounds as sole carbon and energy source has recently been reported. The pathways of metabolism of MTBE involve the initial attack by a monooxygenase. In several cases, the enzyme was characterized as a cytochrome P-450. After oxygenation, the release of a C₁-unit as formaldehyde or formate leads to the production of TBA, which can be converted to 2-hydroxyisobutyric acid and further metabolized. Developments in microbiology make biological treatment of water contaminated with MTBE and other oxygenates an attractive possibility. Work concerning ex situ treatment in biofilters by consortia and by pure strains, and involving or not cometabolism, is under way. Furthermore, the development of in situ treatment processes is a promisinggoal.

Flow cytometry in biotechnology by Marco Rieseberg; Cornelia Kasper; Kenneth F. Reardon; Thomas Scheper (pp. 350-360).

Flow cytometry is a general method for rapidly analyzing large numbers of cells individually using light-scattering, fluorescence, and absorbence measurements. The power of this method lies both in the wide range of cellular parameters that can be determined and in the ability to obtain information on how these parameters are distributed in the cell population. Flow cytometric assays have been developed to determine both cellular characteristics such as size, membrane potential, and intracellular pH, and the levels of cellular components such as DNA, protein, surface receptors, and calcium. Measurements that reveal the distribution of these parameters in cell populations are important for biotechnology, because they better describe the population than the average values obtained from traditional techniques. This Mini-Review provides an overview of the principles of flow cytometry, with descriptions of methods used to measure various cellular parameters and examples of the application of flow cytometry in biotechnology. Finally, a discussion of the challenges and limitations of the method is presented along with a future outlook.

Factors influencing the internalization of Staphylococcus aureus and impacts on the course of infections in humans by E. Alexander; M. Hudson (pp. 361-366).

Staphylococcus aureus is the primary etiological agent of several human diseases. S. aureus has classically been considered an extracellular pathogen; however, recent evidence indicates that S. aureus invades and persists in non-professional phagocytes. Experiments demonstrate that actin microfilaments, microtubules, receptor-mediated endocytosis, and protein tyrosine kinases play important roles in the uptake of S. aureus. Fibronectin-binding proteins and β₁-integrins are implicated as critical cell surface molecules associated with internalization of S. aureus by non-phagocytic cells. Following invasion of eukaryotic cells, S. aureus induces the release of cytokines that have the potential to exacerbate disease and induce apoptosis. Finally, S. aureus has the ability to persist inside host cells as small colony variants, a phenotype associated with persistent and recurrent infections.

Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products by E. Costa; N. Teixidó; J. Usall; E. Atarés; I. Viñas (pp. 367-371).

The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strain CPA-2 of the biocontrol agent Pantoea agglomerans and minimum cost of media, whilst maintaining biocontrol efficacy. To reduce the cost of media, commercial products and by-products were tested. P. agglomerans can be produced using a combination of nitrogen sources such as yeast extract (5 g l^–1) and dry beer yeast (10 g l^–1) with inexpensive carbohydrates such as sucrose (10 g l^–1) and molasses (20 g l^–1), respectively, maintaining the efficacy of the biocontrol agent against Penicillium digitatum and P. italicum on oranges. The results obtained in this study could be used to provide a reliable basis for a scale-up of this fermentation process to an industrial level.

Production of the fungal biocontrol agent Ulocladium atrum by submerged fermentation: accumulation of endogenous reserves and shelf-life studies by S. Frey; N. Magan (pp. 372-377).

A method was developed for the induction of submerged conidiation of Ulocladium atrum Preuss (isolate 385) for the first time, using an oatmeal extract broth. Two inoculum types were produced by this process: spores and mycelial fragments. Spore production was stimulated by reducing the broth water potential (Ψ) to –2.1 MPa and adding 20 mM calcium chloride. In contrast, mycelial fragments were dominant at –7.0 MPa Ψ. Maximum total inoculum (mycelial fragments and conidia) yields were approximately 2×10⁷ ml^–1 after 9 days incubation at 25 °C at 100 rpm. Biomass from liquid cultures responded to water-stress by accumulating increased concentrations of endogenous sugar alcohols (polyols), particularly glycerol. Long-term shelf-life studies showed that submerged inoculum from cultures subjected to an intermediate water-stress (–2.1 MPa Ψ) and containing enhanced levels of glycerol (>300 mg g^–1 freeze-dried material) retained viability significantly better (P<0.05) than that from unstressed cultures, when assessed on agar with fully available water. This level of viability was comparable to that of aerial U. atrum spores from a 4-week solid-substrate fermentation on oat grains. However, in contrast to aerial spores, the ability of submerged biomass to germinate in drier conditions declined significantly after 6 months.

Optimization of enterocin P production by batch fermentation of Enterococcus faecium P13 at constant pH by C. Herranz; J. Martínez; J. Rodríguez; P. Hernández; L. Cintas (pp. 378-383).

The influence of pH on growth, enterocin P production and glucose consumption by Enterococcus faecium P13 was studied during anaerobic batch fermentation in MRS broth at 32 °C in a fermentor. Growth and glucose consumption were maximal at pH 7.0. Enterocin P production displayed primary metabolite kinetics and was strongly dependent on pH. A maximum antimicrobial activity of 1,949 bacteriocin units (BU) ml^–1 was obtained at pH 6.0, which represented a four-fold increase compared with the antimicrobial activity obtained without pH regulation. The pH exerted a marked effect on the decrease in bacteriocin activity,with the decrease being maximal at pH 7.0. In this report, we propose models for the growth of E. faecium P13 as well as enterocin P production and inactivation. Enterocin P production decreased when potentially stress-inducing compounds (NaCl or ethanol) were included in the growth medium.

Shear stress enhances microcin B17 production in a rotating wall bioreactor, but ethanol stress does not by Q. Gao; A. Fang; D. Pierson; S. Mishra; A. Demain (pp. 384-387).

Stress, including that caused by ethanol, has been shown to induce or promote secondary metabolism in a number of microbial systems. Rotating-wall bioreactors provide a low stress and simulated microgravity environment which, however, supports only poor production of microcin B17 by Escherichia coli ZK650, as compared to production in agitated flasks. We wondered whether the poor production is due to the low level of stress and whether increasing stress in the bioreactors would raise the amount of microcin B17 formed. We found that applying shear stress by addition of a single Teflon bead to a rotating wall bioreactor improved microcin B17 production. By contrast, addition of various concentrations of ethanol to such bioreactors (or to shaken flasks) failed to increase microcin B17 production. Ethanol stress merely decreased production and, at higher concentrations, inhibited growth. Interestingly, cells growing in the bioreactor were much more resistant to the growth-inhibitory and production-inhibitory effects of ethanol than cells growing in shaken flasks.

Characterization of native glutamate dehydrogenase from an aerobic hyperthermophilic archaeon Aeropyrum pernix K1 by I. Helianti; Y. Morita; A. Yamamura; Y. Murakami; K. Yokoyama; E. Tamiya (pp. 388-394).

Glutamate dehydrogenase (GDH) was purified and characterized from an aerobic hyperthermophilic archaeon Aeropyrum pernix (A. pernix) K1. The enzyme has a hexameric structure with a native molecular mass of about 285±15 kDa. It was specific for NADP and thermostable (74% activity was remained after 5 h incubation at 100 °C). The activity of the enzyme increased in the presence of polar water-miscible organic solvents such as acetonitrile, methanol, and ethanol. The N-terminal sequence of GDH is Met-Gln-Pro-Thr-Asp-Pro-Leu-Glu-Glu-Ala. This sequence, except for the methionine, corresponds to amino acids 7–15 of the open reading frame (ORF) encoding the predicted GDH (ORF APE 1386). In the ORF nucleotide sequence, the codon TTG appears at the position of the methionine, suggesting that the leucine codon might be recognized as an initiation codon and translated to methionine in A. pernix GDH.

Chitinase from Bacillus thuringiensis subsp. pakistani by S. Thamthiankul; S. Suan-Ngay; S. Tantimavanich; W. Panbangred (pp. 395-401).

The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3–18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.

Isolation and characterization of a cell-associated protein of Bacillus pumilus PH-01 by H.-B. Hong; Y.-S. Chang; S.-D. Choi; I.-H. Nam; Y.-E. Lee (pp. 402-405).

A cell-associated protein released from Bacillus pumilus PH-01 showed an affinity for some dioxins, like 1,2,3,4-tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4-tetrachlorodibenzofuran (TCDF), and the concentration of the protein increased when B. pumilus PH-01 was boiled in minimal salts medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-mass spectrometry revealed that the boiled culture supernatant contained a major protein with a molecular mass of 5,313.4 Da. The adsorption behavior of the protein for 1,2,3,4-TCDD and 1,2,3,4-TCDF was examined by digesting it with proteinase K and trypsin, showing that the proteolyzed protein lost the ability to adsorb the compounds. The amino acid sequence of the protein was determined by automated Edman degradation and tandem mass spectrometry. A search of the protein databases showed no existence of proteins with an homologous sequence.

Synthesis of β-D-glucosyl- and β-D-fucosyl-glucoses using β-glycosidase from Thermus thermophilus by L. Fourage; B. Colas (pp. 406-410).

The thermostable β-glycosidase from Thermus thermophilus, cloned and overexpressed in Escherichia coli was used to catalyze the transfer of β-D-glucosyl and β-D-fucosyl from the corresponding p-nitrophenyl-β-D-glycopyranosides to a hydroxyl group of glucose in the synthesis of β-D-glucosyl-D-glucopyranoses and β-D-fucosyl-D-glucopyranoses. The yields in disaccharides produced under conditions of non-initial velocity were very attractive and the formation of the β(1–3) linked disaccharides was largely favored. The enzyme could constitute a valuable biocatalyst for the synthesis of disaccharides involving such structures as, for example, Bifidus factors.

Electroinduced extraction of β-galactosidase from Kluyveromyces lactis by V. Ganeva; B. Galutzov; N. Eynard; J. Teissié (pp. 411-413).

A new methodology for the extraction of β-galactosidase from the yeast Kluyveromyces lactis was obtained by electropulsation. The application of a series of electric pulses (2 ms duration, 1 Hz frequency, and 4–4.5 kV/cm field strength) to fresh cells suspended in deionized water, followed by incubation in PBS, led to a spontaneous slow release of enzyme at a yield of 75–80% without any further treatment. Most of the enzyme was extracted within 8 h after electropulsation. This release was dependent on the growth phase. The specific activity of β-galactosidase in the supernatant of pulsed cells was higher by a factor of 1.5–1.7 in comparison with crude extract.

Disruption of a gene encoding a putative γ-butyrolactone-binding protein in Streptomyces tendae affects nikkomycin production by P. Engel; L. Scharfenstein; J. Dyer; J. Cary (pp. 414-419).

A 2.6-kb BamHI fragment from the genome of the wild-type, nikkomycin-producing strain of Streptomyces tendae ATCC 31160 was cloned and sequenced. This 2.6-kb BamHI fragment corresponds to the DNA site where transposon Tn4560 had inserted to create a nikkomycin-nonproducing mutant. A possible ORF of 660 nucleotides was found in this 2.6-kb BamHI fragment, in which the third base of each codon was either G or C in 92% of the codons. The deduced amino acid sequence coded by this ORF (TarA, tendae autoregulator receptor) shows strong homology with several γ-butyrolactone-binding proteins that negatively regulate antibiotic production in other streptomycetes and have a helix-turn-helix DNA-binding motif. A portion (179 nucleotides) of tarA that encodes the helix-turn-helix motif was replaced with ermE, and wild-type S. tendae was transformed with this construct borne in pDH5, a gene-disruption vector. Southern hybridization indicated that ermE had inserted in the 2.6-kb BamHI region in one isolate that is erythromycin resistant. Northern hybridization indicated that tarA disruption significantly increased the amount of disrupted-tarA mRNA. This suggests that TarA negatively regulates its own synthesis. Nikkomycin production by the tarA disruptant was delayed but reached the wild-type level after longer incubation in production medium.

Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast by A. Geerlings; F. Redondo; A. Contin; J. Memelink; R. van der Heijden; R. Verpoorte (pp. 420-424).

A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine β-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.

Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene by C. Wang; A. Lum; S. Ozuna; D. Clark; J. Keasling (pp. 425-430).

The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.

Heterologous expression of the Bacillus pumilus endo-β-xylanase (xynA) gene in the yeast Saccharomyces cerevisiae by Filip Nuyens; Willem H. van Zyl; Dirk Iserentant; Hubert Verachtert; Chris Michiels (pp. 431-434).

The endo-β-xylanase-encoding gene (xynA) of Bacillus pumilus PLS was isolated from a genomic DNA library and the open reading frame (ORF) was inserted in expression vectors for the yeast Saccharomyces cerevisiae. Plasmid pFN3 harboured the xynA ORF fused to the yeast mating pheromone alpha-factor signal sequence (MFα1 S) under the control of the alcohol dehydrogenase II gene promotor (ADH2 P) and terminator (ADH2 T) sequences. In plasmid pFN4, the MFα1 S-xynA ORF was brought under the control of the phosphoglycerate kinase I gene promotor (PGK1 P) and terminator (PGK1 T) sequences. Autoselective, recombinant S. cerevisiae [fur1::LEU2] strains bearing pFN3 or pFN4 secreted functional endo-β-xylanase when grown in complex medium. Enzymatic activities in the culture supernatants reached maximum levels of 8.5 nkat/ml and 4.5 nkat/ml, respectively. The temperature and pH optimum for both the bacterial and the recombinant xylanase were 58 °C and pH 6.2.

Detection of Xanthomonas oryzae pv. oryzae in artificially inoculated and naturally infected rice seeds and plants by molecular techniques by N. Sakthivel; C. Mortensen; S. Mathur (pp. 435-441).

A polymerase chain reaction (PCR) technique was developed for detecting the presence of Xanthomonas oryzae pv. oryzae, the bacterial leaf blight (BLB) pathogen in rice seed and for studying the transmission of this bacterium from seed to plant. Primers TXT and TXT4R from an insertion sequence (IS1113) of the pathogen were used to amplify a 964-bp DNA fragment. A combined biological and enzymatic amplification (BIO-PCR) technique was used to detect the pathogen in naturally infected seed. The level of detection of TXT and TXT4R primers was 55 fg DNA of X. o. pv. oryzae, which is roughly the equivalent of seven cells (and four cells in pure culture suspension) of X. o. pv. oryzae. Hybridization of IS1113 with the amplified DNA fragment in Southern blot analysis confirmed that the 964-bp DNA fragment was amplified from X. o. pv. oryzae. The presence of the IS1113 element in strains of X. o. pv. oryzae from 16 rice-growing countries was confirmed by DNA dot blot analysis. X. o. pv. oryzae was detected from the seed washes and DNA extracted from the seed washes of naturally infected seeds of cvs Jaya and TN1. When stored at 4 °C, the pathogen was recovered up to 4 months and 9 months from naturally infected seeds of cvs Jaya and TN1, respectively. The BLB bacterium was also detected in seedlings, mature plants and seeds collected from plants raised from naturally infected seeds.

Cloning and sequencing of a gene encoding a novel salt stress-induced membrane protein from Rhodobacter sphaeroides f. sp. denitrificans by XY. Xu; H. Kadokura; A. Okubo; K. Kitamoto; S. Yamazaki (pp. 442-447).

The gene encoding a membrane protein, SspA, induced under salt stress conditions was cloned and sequenced from a photosynthetic bacterium, Rhodobacter sphaeroides f. sp. denitrificans IL106. A single open reading frame consisting of 972 base pairs that encoded a polypeptide composed of a signal peptide of 24 amino acids and a mature protein of 300 amino acids (M _r 33,386) was found. A database search failed to detect any highly homologous sequences, indicating that SspA is a novel protein. The protein was present in the outer membrane as a transmembrane protein and was specifically induced by salt stress, but not by heat shock.

Isolation and characterization of a bacterium that produces hydrocarbons extracellularly which are equivalent to light oil by M.-O. Park; M. Tanabe; K. Hirata; K. Miyamoto (pp. 448-452).

A halotorelant bacterial strain that produces a significant amount of lipids from short-chain fatty acids was isolated from the sludge of a sewage disposal plant. This strain displayed a significant extracellular accumulation of lipids. The yield of lipids including hydrocarbons was highest (120% of cell dry weight) at the end of the linear growth phase. Fractionation of the lipids using thin-layer chromatography and subsequent gas chromatography showed that hydrocarbons were also obtained following an increase in total lipids. Their yield was the highest (50% of cell dry weight) in the linear growth phase. Additional analysis using infrared absorption spectrum and gas chromatography-mass spectrometry showed that the hydrocarbon fraction was composed of alkanes, such as C₁₅H₃₂, C₁₈H₃₈, C₂₁H₄₄, C₂₂H₄₆ and C₂₄H₅₀. Homology analysis of the 16s rDNA sequence as well as studies of the morphological and physiological characteristics indicated that the bacterium is a strain of Vibrio furnissii.

Biological production of optically active muconolactones by Rhodococcus rhodochrous by C.-J. Cha (pp. 453-457).

Optically active (-)-3-methylmuconolactone was biologically produced using a mutant strain of Rhodococcus rhodochrous N75 that is capable of metabolizing 4-methylcatechol via a modified ortho-cleavage pathway. The mutant strain (CJ30) was prepared by mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine and found to be blocked in the degradation of 3-methylmuconolactone. Cells of the mutant CJ30, which had been previously grown on yeast extract and induced with p-toluate, transformed p-toluate (11.5 mM) to optically active (-)-3-methylmuconolactone with a yield of 53%. The structure of 3-methylmuconolactone was confirmed by NMR spectroscopy and mass spectrometry. Cell-free extracts of R. rhodochrous N75 also transformed a range of 4-alkylcatechols, such as 4-ethylcatechol, 4-iso-propylcatechol, and 4-tert-butylcatechol, to the corresponding 4-alkyl-substituted muconolactones.

A cytochrome P450 and a ferredoxin isolated from Mycobacterium sp. strain HE5 after growth on morpholine by B. Sielaff; J. Andreesen; T. Schräder (pp. 458-464).

A cytochrome P450 and an iron-sulfur protein, whose expression was specifically induced during growth of Mycobacterium sp. strain HE5 on morpholine as the sole source of carbon, nitrogen, and energy were purified to apparent homogeneity. Due to the lack of enzymatic activity, carbon monoxide difference spectra and determination of the acid-labile sulfur, respectively, were used to detect the proteins during purification. The cytochrome P450, designated P450_mor, was characterized as a monomer with an apparent molecular mass of 44.7 kDa. The amino acid sequence of an internal peptide comprising 19 amino acids was identical to the sequence derived from a gene encoding a cytochrome P450 from Mycobacterium smegmatis mc²155 suggested to be involved in the utilization of piperidine and pyrrolidine. The iron-sulfur protein was characterized as a ferredoxin exhibiting a molecular mass of 6.8 kDa and named Fd_mor. An identity of 48–77% was obtained for the 30 N-terminal amino acids of Fd_mor and the corresponding sequences of different 3Fe-4S-ferredoxins known to be involved in P450-dependent reactions. From these data we concluded that growth of Mycobacterium sp. strain HE5 on morpholine led to the expression of a cytochrome P450-dependent monooxygenase system composed of at least two different proteins.

Novel glucoamylase-type enzymes from Thermoactinomyces vulgaris and Methanococcus jannaschii whose genes are found in the flanking region of the α-amylase genes by R. Uotsu-Tomita; T. Tonozuka; H. Sakai; Y. Sakano (pp. 465-473).

A region downstream of the gene for pullulan-hydrolyzing α-amylase, TVA II, of Thermoactinomyces vulgaris R-47 was sequenced, and an open reading frame encoding an enzyme homologous to glucoamylase was found. The nucleotide sequence of this enzyme, designated TGA, consists of 1,953 base pairs corresponding to a protein of 651 amino acid residues. The TGA gene was subcloned and expressed in Escherichia coli. Enzymatic analyses showed that, like other glucoamylases, TGA produced β-D-glucose from its substrate. However, TGA hydrolyzed maltooligosaccharides such as maltotetraose and maltose more efficiently than starch, while fungal glucoamylases preferred starch to maltooligosaccharides. The primary structure of TGA resembled a putative glucoamylase from the hyperthermophilic archaeon Methanococcus jannaschii (MGA), while homologies between TGA and the fungal glucoamylases were low. The enzymatic properties of recombinant MGA produced in E. coli cells were similar to those of TGA. These findings indicate that TGA and MGA are novel glucoamylase-type enzymes with oligosaccaharide-metabolizing activity.

The fungus Pestalotiopsis guepini as a model for biotransformation of ciprofloxacin and norfloxacin by I. Parshikov; T. Heinze; J. Moody; J. Freeman; A. Williams; J. Sutherland (pp. 474-477).

The metabolism of the fluoroquinolone drugs ciprofloxacin and norfloxacin by Pestalotiopsis guepini strain P-8 was investigated. Cultures were grown at 28 °C in sucrose/peptone broth for 18 days after dosing with ciprofloxacin (300 µM) or norfloxacin (313 µM). Four major metabolites were produced from each drug; and these were purified by high-performance liquid chromatography and identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Ciprofloxacin metabolites included N-acetylciprofloxacin (52.0%), desethylene-N-acetylciprofloxacin (9.2%), N-formylciprofloxacin (4.2%), and 7-amino-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.3%). Norfloxacin metabolites included N-acetylnorfloxacin (55.4%

Keywords: ), desethylene-N-acetylnorfloxacin (8.8%), N-formylnorfloxacin (3.6%), and 7-amino-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.1%). N-Formylciprofloxacin and the four transformation products from norfloxacin are all known to be mammalian metabolites.

Repression of fatty-acyl-CoA oxidase-encoding gene expression is not necessarily a determinant of high-level production of dicarboxylic acids in industrial dicarboxylic-acid-producing Candida tropicalis by A. Hara; M. Ueda; T. Matsui; M. Arie; H. Saeki; H. Matsuda; K. Furuhashi; T. Kanai; A. Tanaka (pp. 478-485).

The synthesis of dicarboxylic acids (DCAs) in Candida tropicalis is thought to be induced by a decrease in fatty acyl-CoA-oxidase activity. However, in the present study we demonstrate that repression of the POX4 gene, encoding fatty acyl-CoA oxidase, does not directly lead to high-level production of DCAs. No fatty acyl-CoA-oxidase activity was detected if the POX4 gene of C. tropicalis strain 1098 (wild-type strain) was disrupted. Furthermore, introduction of the POX4 gene from C. tropicalis strain M1210A3, which is a mutant derived from strain 1098 and is used as an industrial DCA-producing strain, still exhibited low-level fatty acyl-CoA-oxidase activity. Nevertheless, production of DCA was not observed in either case. Furthermore, the increase in acyl-CoA-oxidase activity by expression of the POX4 gene in strain M1210A3 did not reduce high-level production of DCA. These results suggest that alterations in acyl-CoA-oxidase activity are not necessarily related to production of DCA in industrial DCA-producing C. tropicalis M1210A3.

Mineralization and co-metabolic degradation of phenoxyalkanoic acid herbicides by a pure bacterial culture isolated from an aquifer by P. Mai; O. Stig Jacobsen; J. Aamand (pp. 486-490).

A mecoprop [(±)-2-(4-chloro-2-methylphenoxy)propionic acid; MCPP]-degrading bacterium identified as Stenotrophomonas maltophilia PM was isolated from a Danish aquifer. Besides mecoprop, the bacterium was also able to degrade MCPA [(4-chloro-2-methylphenoxy)acetic acid)], MCPB [(4-chloro-2-methylphenoxy)butyric acid], 4-CPA [(4-chlorophenoxy)acetic acid], 2, 4-D [(2, 4-dichlorophenoxy)acetic acid], 2, 4-DP [(±)-2-(2, 4-dichlorophenoxy)propionic acid] and 2, 4-DB [(2, 4-dichlorophenoxy)butyric acid]. The bacterium was able to grow using these individual phenoxyalkanoic acids as the sole source of carbon and energy. In addition, it was able to co-metabolically degrade the phenoxyalkanoic acid 2, 4, 5-T [(2, 4, 5-trichlorophenoxy)acetic acid)] in the presence of mecoprop. At high 2, 4, 5-T concentrations (100 and 52 mg/l), however, only partial degradation of both mecoprop and 2, 4, 5-T was obtained, thus indicating the production of toxic metabolites. Bacterial yields were highest when grown on the monochlorinated phenoxyalkanoic acids as compared to the dichlorinated analogues, an exception being growth on 4CPA, which resulted in the lowest yield at all. Using [ring-U-¹⁴C]-labeled herbicides it was shown that the lower yield on 2, 4-D than on mecoprop was accompanied by greater CO₂ generation, thus indicating that less energy is available from the complete oxidation of the dichlorinated phenoxyalkanoic acids than the monochlorinated analogues.

Transformation of 2,2′-dichlorodiisopropyl ether in mixed and pure culture by R. Hauck; L. Adrian; P. Wendler; M. Amidjojo; W. Hegemann; H. Görisch (pp. 491-495).

An aerobic enrichment culture derived from a groundwater contaminated with organic and chloroorganic compounds was adapted to the transformation of 2,2′-dichlorodiisopropyl ether (DDE) in a continuous fixed-bed bioreactor. Continuous DDE removal efficiencies over 90% were achieved with a model water containing 3.3 mM methanol as co-substrate at DDE loading rates of up to 150 µmol l^–1 day^–1 with a hydraulic retention time of 24 h. In batch cultures, a stoichiometric release of 2 µmol chloride per µmol DDE transformed was observed. From the mixed culture, a strain was isolated that is able to grow on DDE as the sole energy and carbon source, tolerating DDE concentrations of up to 1 mM. Based on 16S rRNA sequencing, the strain is affiliated with the genus Rhodococcus.

The effect of osmotic stress on the production of nukacin ISK-1 from Staphylococcus warneri ISK-1 by T. Sashihara; M. Dan; H. Kimura; H. Matsusaki; K. Sonomoto; A. Ishizaki (pp. 496-501).

The effects of several additives on the production of a lantibiotic, nukacin ISK-1, from Staphylococcus warneri ISK-1, in batch fermentation were studied. NaCl, KCl and sorbitol stimulated nukacin ISK-1 production. The addition of 1.4 M NaCl increased nukacin ISK-1 activity 1.5-fold over the control, while cell growth and glucose consumption were inhibited. Nukacin ISK-1 production increased with increasing osmolarity of the medium up to about 3 osmol/kg; however, further increases in osmolarity diminished productivity, irrespective of the kind of additive. Northern blot analysis showed that transcription of the nukacin ISK-1 structural gene (nukA) was activated in the presence of 1.4 M NaCl. These data indicate that the stimulation effect was due to osmotic stress, which acted, at least in part, at the transcriptional level on the nukA gene.

An antibiotic with activity against gram-positive bacteria from the gentamicin-producing strain of Micromonospora purpurea by K. Rusnak; J. Troyanovich; R. Mierzwa; M. Chu; M. Patel; M. Weinstein (pp. 502-503).

A mixture of polycyclic aromatic compounds with activity against gram-positive bacteria was isolated from a gentamicin-producing species of Micromonospora.

Evaluation of nitric oxide production by lactobacilli by J. Xu; W. Verstraete (pp. 504-507).

Six strains of Lactobacillus fermentum and Lactobacillus plantarum were investigated for nitric oxide (NO) production. First, the potential presence of NO synthase was examined. None of the strains of L. fermentum and L. plantarum examined produced NO from L-arginine under aerobic conditions. Interestingly, all L. fermentum strains expressed strong L-arginine deiminase activity. All L. fermentum strains produced NO in MRS broth, but the NO was found to be chemically derived from nitrite, which was produced by L. fermentum from nitrate present in the medium. Indeed all L. fermentum strains express nitrate reductase under anaerobic conditions. Moreover, one strain, L. fermentum LF1, had nitrate reductase activity under aerobic conditions. It was also found that L. fermentum strains JCM1173 and LF1 possessed ammonifying nitrite reductase. The latter strain also had denitrifying nitrite reductase activity at neutral pH under both anaerobic and aerobic conditions. The LF1 strain is thus capable of biochemically converting nitrate to NO. NO and nitrite produced from nitrate by lactobacilli may constitute a potential antimicrobial mechanism.

The effect of lithium chloride on the biooxidation of aqueous methanol/acetone mixtures by M. O'Brien; G. Hamer (pp. 508-512).

Lithium chloride, more specifically the lithium cation, has been implicated in interference in biological systems. In the case of Escherichia coli, interference involves the Na⁺(Li⁺)/H⁺ antiporter transport system. The study reported here concerns the effects of LiCl on a mixed enrichment culture that is able to biodegrade both methanol and acetone under aerobic conditions. The results obtained using unsteady state continuous flow culture techniques demonstrate a significant disruptive effect of LiCl on culture performance. In addition, a reduction in the substrate-based biomass yield coefficient, which is a clear advantage as far as biotreatment process performance is concerned, also occurs. The ultimate fate of the LiCl was not determined.

Coupling effects of osmotic pressure and temperature on the viability of Saccharomyces cerevisiae by L. Beney; P. Marechal; P. Gervais (pp. 513-516).

The osmotic tolerance of cells of Saccharomyces cerevisiae as a function of glycerol concentration and temperature has been investigated. Results show that under isothermal conditions (25 °C) cells are resistant (94% viability) to hyperosmotic treatment at 49.2 MPa. A thigher osmotic pressure, cell viability decreases to 25% at 99 MPa. Yeast resistance to high osmotic stress (99 Mpa) is enhanced at low temperatures (5–11 °C). Therefore, the temperature at which hyperosmotic pressure is achieved greatly affects cell viability. These results suggest that temperature control is a suitable means of enhancing cell survival in response to osmotic dehydration.

κ-carrageenan/gelatin gel beads for the co-immobilization of aerobic and anaerobic microbial communities degrading 2,4,6-trichlorophenol under air-limited conditions by H. Gardin; A. Pauss (pp. 517-523).

Alginate and κ-carrageenan gels were tested as bead materials for the co-immobilization of anaerobic and aerobic microorganisms for the mineralization of 2,4,6-trichlorophenol under air-limited conditions. Chemical, mechanical and thermal culture constraints were pre-defined and the gel resistances were established. Alginate was quickly eliminated because of its chemical instability in the culture media. In anaerobic conditions, the microorganisms transformed the substrates into CH₄and CO₂. The κ-carrageenan gel did not enable these gases to diffuse. They remained as bubbles in the core of the beads and made the beads float. Gelatin was added to κ-carrageenan in order to change the carrier properties. No biogas bubbles appeared in the bead core during the cultures in anaerobic conditions and the beads reacted well to the culture conditions in the reactor. The co-immobilization of the anaerobic and aerobic microbial communities was successfully performed with the κ-carrageenan/gelatin gel (2% (w/w) of each polymer). The biological activities, measured by the impedancemetry technique, were preserved in the beads.

Degradation of 2,4,6-trichlorophenol (2,4,6-TCP) by co-immobilization of anaerobic and aerobic microbial communities in an upflow reactor under air-limited conditions by H. Gardin; J. Lebeault; A. Pauss (pp. 524-530).

The co-immobilization and the culture of anaerobic and aerobic communities was tested for the mineralization of 2,4,6-trichlorophenol (2,4,6-TCP). At first, the anaerobic microorganisms (aggregated into granules) were cultivated in an upflow anaerobic sludge blanket (UASB) reactor, in a continuous mode, with glucose, propionate, acetate (COD loading rate = 0.5–2.0 g COD/l per day, ratio 1:1:1) and 2,4,6-TCP (2,4,6-TCP loading rate = 25–278 µmol/l per day) as substrates. 2,4,6-TCP was degraded into 2,4-DCP and 4-CP, but it was not mineralized because of the low degradation rates of 4-CP. Furthermore, the highest loading rates of 2,4,6-TCP (>126 µmol/l per day) caused the inhibition of the strains degrading the propionate. The granules were therefore tested in association with the aerobic community. They were immobilized in κ-carrageenan/gelatin [2% (w/w) of each polymer] gel beads and cultivated in a reactor, on their own (to test the influence of the gel), and then with the aerobic community, under anaerobic and air-limited conditions, respectively. The results showed that (1) the gel did not influence the activity of the granules, (2) the anaerobic and aerobic communities could be easily co-immobilized in gel beads and cultivated in a reactor, (3) the mineralization of 2,4,6-TCP (2,4,6-TCP loading rate = 10–506 µmol/l per day), its intermediates of degradation and the other substrates [glucose + acetate + propionate (ratio 1:1:1) = COD loading rate = 500 mg COD/l per day] could be obtained under air-limited conditions if the culture parameters were strictly controlled [airflow = 36–48 vvd (volume of air/volume of liquid in the reactor per day), pH value at around 7.5]. Finally, the gel did not retain its structure during the whole culture (263 days) in the air-limited reactor, but the anaerobic and aerobic communities retained their activities and worked together for the mineralization.

The impact of fermentative organisms on carbon flow in methanogenic systems under constant low-substrate conditions by S. Dollhopf; S. Hashsham; F. Dazzo; R. Hickey; C. Criddle; J. Tiedje (pp. 531-538).

We compared carbon flow under constant low-substrate conditions (below 20 µM glucose in situ) in laboratory-scale glucose-fed methanogenic bioreactors containing two very different microbial communities that removed chemical oxygen demand at similar rates. One community contained approximately equal proportions of spiral and cocci morphologies, while the other community was dominated by cocci. In the former bioreactor, over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Spirochaetaceae-related sequences, while in the latter bioreactor over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Streptococcus-related sequences. Carbon flow was assessed by measuring ¹⁴C-labeled metabolites derived from a feeding of [U-¹⁴C]glucose that did not alter the concentration of glucose in the bioreactors. Acetate and ethanol were detected in the Spirochaetaceae-dominated reactor, whereas acetate and propionate were detected in the Streptococcus-dominated reactor. A spirochete isolated from a Spirochaetaceae-dominated reactor fermented glucose to acetate, ethanol, and small amounts of lactate. Maximum substrate utilization assays carried out on fluid from the same reactor indicated that acetate and ethanol were rapidly utilized by this community. These data indicate that an acetate- and ethanol-based food chain was present in the Spirochaetaceae-dominated bioreactor, while the typical acetate- and propionate-based food chain was prevalent in the Streptococcus-dominated bioreactor.

Degradation of phenanthrene-analogue azaarenes by Mycobacterium gilvum strain LB307T under aerobic conditions by P. Willumsen; J. Nielsen; U. Karlson (pp. 539-544).

A polycyclic aromatic hydrocarbon degrading Mycobacterium gilvum, strain LB307T, was able to degrade the azaarenes 5,6-benzoquinoline, 7,8-benzoquinoline, and phenanthridine (nitrogen-containing heterocyclic aromatic hydrocarbons) under aerobic conditions. The strain was able to use 5,6-benzoquinoline as sole sources of carbon, nitrogen, and energy. However, inhibition of degradation and growth was observed with increasing substrate concentration. During degradation, metabolites built up transiently. One of the metabolites detected during 5,6-benzoquinoline degradation is suggested to be 2-oxo-5,6-benzoquinoline. This is the first report on bacterial degradation of phenanthrene-analogue azaarenes.

Tetrachloroethylene, trichloroethylene, and chlorinated phenols induce toluene-o-xylene monooxygenase activity in Pseudomonas stutzeri OX1 by D. Ryoo; H. Shim; F. Arenghi; P. Barbieri; T. Wood (pp. 545-549).

Pseudomonas stutzeri OX1 naphthalene-oxidation activity is induced 3.0-fold by tetrachloroethylene (PCE) and 3.1-fold by trichloroethylene (TCE) at 100 µM. With the mutant P. stutzeri M1, which does not express toluene-o-xylene monooxygenase (ToMO, product of the tou operon), no naphthalene-oxidation activity induction by PCE and TCE was found; hence, PCE and TCE induce ToMO of P. stutzeri OX1. The chlorinated phenols 2-, 3-, and 4-chlorophenol induced ToMO expression 0.58-, 0.23- and 0.37-fold, respectively, compared to the direct inducer of the pathway, o-cresol. Using P. putida PaW340 (pPP4062, pFP3028), which has the tou promoter fused to the reporter catechol-2,3-dioxygenase, and the regulator gene touR, it was determined that the tou promoter was induced directly 5.7-, 7.1-, and 5.1-fold for 2-, 3-, and 4-chlorophenol, respectively (compared to an 8.8-fold induction with o-cresol). In addition, it was found that TCE and PCE do not directly induce the tou pathway and that components other than the tou structural and regulatory genes are necessary for induction. Gas chromatography results also showed that 100 µM TCE induced its own degradation (8–9%) in 16 h in P. stutzeri OX1, and all of the stoichiometric chloride from the degraded TCE was detected in solution.

Mediated microbial biosensor using a novel yeast strain for wastewater BOD measurement by S. Trosok; B. Driscoll; J. Luong (pp. 550-554).

Two new yeast strains (SPT1 and SPT2) were isolated and immobilized on glassy carbon electrodes to form microbial biosensors for estimation of biochemical oxygen demand (BOD). Ferricyanide was proven to be the most efficient mediator to shuttle electrons from the redox center of reduced microbial enzymes to the electrode in the presence of excess glucose/glutamic acid (GGA). With a 3-fold greater metabolic assimilation capability and greater responses to various effluent samples, SPT1 was selected for sensor-BOD measurements. BOD estimations for the GGA standard resulted in an extended linear range: 2–100 mg/l. Response reproducibility was ±10% for a GGA standard containing 10 mg BOD/l. For analysis of pulp mill effluents, the BOD detection limit was 2 mg/l with a response time of 5 min.

Enhancing pyrene mineralization in contaminated soil by the addition of humic acids or composted contaminated soil by A. Haderlein; R. Legros; B. Ramsay (pp. 555-559).

The addition of composted PAH-contaminated soil to PAH-contaminated soil spiked with ¹⁴C-labeled pyrene resulted in rapid mineralization of pyrene (more than 57% after 21 days compared with 3.4% in unamended soil). The addition of the humic acid fraction of the composted soil also increased the mineralization potential of the soil significantly, but to a lesser extent (37.5% mineralization after 106 days compared with 20.6% in unamended soil). Increasing the humic acid concentration increased mineralization up to a maximum of more than three times the unamended rate, after which the rate of pyrene mineralization decreased, possibly due to inhibitory pH or concentrations of salts. The amendment of PAH-contaminated soil with materials containing humic acids or humic acid extracts is suggested as a method of bioremediation.

Ferrous sulphate oxidation using Thiobacillus ferrooxidans cells immobilised on sand for the purpose of treating acid mine-drainage by T. Wood; K. Murray; J. Burgess (pp. 560-566).

Thiobacillus ferrooxidans was immobilised on sand (size 0.85 mm to 1.18 mm) for use in a repeated batch and continuously operated packed-bed bioreactor which has not been previously reported in the literature. Repeated batch operation resulted in the complete oxidation of ferrous to ferric iron. The bacteria were active immediately after 3–4 weeks in a non-aqueous medium; i.e. the sand was allowed to dry out, demonstrating the stability of the system. A lag phase of 28 days was recorded when the sand was stored dried in a sealed container for 16 weeks compared with a lag phase of 13 days for a sample frozen for 18 weeks. After a period of 10 days, continuous operation of the reactor at a dilution rate of 0.64 h^–1 resulted in 95–99% oxidation of ferrous iron or 0.31–0.33 kg m^–3 h^–1. With the use of a scanning electron microscope, images were recorded of Thiobacillus ferrooxidans on sand.

Sections

Personal tools

Applied Microbiology and Biotechnology (v.56, #3-4)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.56, #3-4)