Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Applied Microbiology and Biotechnology (v.59, #1)
Biotechnological production of 2-phenylethanol by M. Etschmann; W. Bluemke; D. Sell; J. Schrader (pp. 1-8).
2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odour. Most of the world's annual production of several thousand tons is synthesised by chemical means but, due to increasing demand for natural flavours, alternative production methods are being sought. Harnessing the Ehrlich pathway of yeasts by bioconversion of L-phenylalanine to 2-PE could be an option, but in situ product removal is necessary due to product inhibition. This review describes the microbial production of 2-PE, and also summarizes the chemical syntheses and the market situation.
Biochemistry and molecular genetics of poly-γ-glutamate synthesis by M. Ashiuchi; H. Misono (pp. 9-14).
Current research into poly-γ-glutamate (PGA) and its biosynthesis is reviewed. In PGA-producing Bacillus subtilis, glutamate racemase supplies abundant DL-glutamate, the substrate for PGA synthesis. The pgsBCA genes of PGA-producing B. subtilis, which encode the membrane-associated PGA synthetase complex PgsBCA, were characterized and the enzyme complex was suggested to be an atypical amide ligase based on its structure and function. A novel reaction mechanism of PGA synthesis is proposed.
Bacterial alkaline proteases: molecular approaches and industrial applications by R. Gupta; Q. Beg; P. Lorenz (pp. 15-32).
Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN′ and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.
Heterotrophic bacteria growing in association with Methylococcus capsulatus (Bath) in a single cell protein production process by Harald Bothe; K. Møller Jensen; A. Mergel; J. Larsen; C. Jørgensen; Hermann Bothe; L. Jørgensen (pp. 33-39).
The methanotrophic bacterium Methylococcus capsulatus (Bath) grows on pure methane. However, in a single cell protein production process using natural gas as methane source, a bacterial consortium is necessary to support growth over longer periods in continuous cultures. In different bioreactors of Norferm Danmark A/S, three bacteria consistently invaded M. capsulatus cultures growing under semi-sterile conditions in continuous culture. These bacteria have now been identified as a not yet described member of the Aneurinibacillus group, a Brevibacillus agri strain, and an acetate-oxidiser of the genus Ralstonia. The physiological roles of these bacteria in the bioreactor culture growing on natural, non-pure methane gas are discussed. The heterotrophic bacteria do not have the genetic capability to produce either the haemolytic enterotoxin complex HBL or non-haemolytic enterotoxin.
The use of co-immobilization of Trichosporon cutaneum and Bacillus licheniformis for a BOD sensor by L. Suriyawattanakul; W. Surareungchai; P. Sritongkam; M. Tanticharoen; K. Kirtikara (pp. 40-44).
The microorganisms Trichosporon cutaneum and Bacillus licheniformis were used to develop a microbial biochemical oxygen demand (BOD) sensor. It was found that T. cutaneum gave a greater response to glucose, whereas B. licheniformis gave a better response to glutamic acid. Hence, co-immobilized T. cutaneum and B. licheniformis were used to construct a glucose and glutamic acid sensor with improved sensitivity and dynamic range. A membrane loading of T. cutaneum at 1.1×108 cells ml–1 cm–2 and B. licheniformis at 2.2×108 cells ml–1 cm–2 gave the optimum result: a linear range up to 40 mg BOD l–1 with a sensitivity of 5.84 nA mg–1 BOD l. The optimized BOD sensor showed operation stability for 58 intermittent batch measurements, with a standard deviation of 0.0362 and a variance of 0.131 nA. The response time of the co-immobilized microbial BOD sensor was within 5–10 min by steady-state measurement and the detection limit was 0.5 mg BOD l–1. The BOD sensor was insensitive to pH in the range of pH 6.8–7.2.
Large scale production and downstream processing of a recombinant porcine parvovirus vaccine by L. Maranga; P. Rueda; A. Antonis; C. Vela; J. Langeveld; J. Casal; M. Carrondo (pp. 45-50).
Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of infection (MOI) strategy was efficiently applied avoiding the use of an extra baculovirus expansion step. The optimal harvest time was defined at 120 h post-infection at the MOI used, with the cell concentration at infection being 1.5×106 cells/ml. An efficient purification scheme using centrifugation, precipitation and ultrafiltration/diafiltration as stepwise unit operations was developed. The global yield of the downstream process was 68%. Baculovirus inactivation with Triton X-100 was successfully integrated into the purification scheme without an increase in the number of process stages. Immunogenicity of the PPV-VLPs tested in guinea pigs was similar to highly purified reference material produced from cells cultured in the presence of serum-containing medium. These results indicate the feasibility of industrial scale production of PPV-VLPs in the baculovirus system, safety of the product, and the potency of the product for vaccine application.
Elicitor-like effects of low-energy ultrasound on plant (Panax ginseng) cells: induction of plant defense responses and secondary metabolite production by J. Wu; L. Lin (pp. 51-57).
In this work we examined the elicitor-like effects of low-energy ultrasound (US) on plant cells with respect to the induction of plant defense responses and secondary metabolite production. Panax ginseng cells in suspension culture were exposed to US (power ≤0.1 W/cm3 at 38.5 kHz fixed frequency) for short periods of time (30 s–6 min). Two early events in plant defense metabolism and signal transduction pathways, the increased cross-membrane ion fluxes (Ca2+ influx and K+ efflux/H+ influx) and the production of active oxygen species (AOS), were detected in sonicated cultures within 2 min after US exposure. These responses could be induced with small doses of US energy, 0.6–0.8 J/cm3, and enhanced by increasing US energy within a non-inhibitory range. US treatment stimulated the synthesis of useful secondary metabolites, saponins of ginseng cells, without causing any net loss of the biomass yield of ginseng cell cultures. These results suggest that US can act as a potent abiotic elicitor to induce the defense responses of plant cells and to stimulate secondary metabolite production in plant cell cultures.
Impact of electrode composition on electricity generation in a single-compartment fuel cell using Shewanella putrefaciens by D. Park; J. Zeikus (pp. 58-61).
The production of electricity by Shewanella putrefaciens in the absence of exogenous electron acceptors was examined in a single compartment fuel cell with different types of electrodes and varying physiological conditions. Electricity production was dependent on anode composition, electron donor type and cell concentration. A maximum current of 2.5 mA and a current density of 10.2 mW/m2 electrode was obtained with a Mn4+ graphite anode, 200 mM sodium lactate and a cell concentration of 3.9 g cell protein/ml. Current production by S. putrefaciens was enhanced 10-fold when an electron mediator (i.e., Mn4+ or neutral red) was incorporated into the graphite anode.
Cometabolic ring fission of dibenzofuran by Gram-negative and Gram-positive biphenyl-utilizing bacteria by M. Stope; D. Becher; E. Hammer; F. Schauer (pp. 62-67).
Thirty-five strains of soil bacteria were grown with biphenyl (BP) and tested for their capacity to cooxidize dibenzofuran (DBF). During metabolism of DBF, the culture medium of 17 strains changed from colorless to orange, indicating a meta-cleavage pathway of DBF degradation. The ring cleavage product of these isolates was shown to be 2-hydroxy-4-(3′-oxo-3′H-benzofuran-2′-yliden)but-2-enoic acid (HOBB). The strain SBUG 271, studied in detail and identified as Rhodococcus erythropolis, degraded DBF via 1,2-dihydroxydibenzofuran. The ensuing meta-cleavage yielded HOBB and salicylic acid. In addition, the four monohydroxylated monomers of DBF and two metabolites, which were not further characterized, were detected. Thus, our results demonstrate that the metabolic mechanism involves lateral dioxygenation of DBF followed by meta-cleavage and occurs in Gram-negative as well as in Gram-positive BP-degrading bacteria.
Inhibition of bacterial α-glucosidases by castanospermine in pure cultures and activated sludge by N. Mustafa; M. Thörn; F. Sörensson (pp. 68-71).
Castanospermine (CAST) is a known and potent inhibitor of various α-glucosidases in eukaryotes. In this work, we elucidated whether CAST could also be used for determining bacterial α-glucosidase activity, when measured with 4-methylumbelliferyl-α-D-glucoside as a substrate, both in a complex bacterial community, in activated sludge and in pure cultures of bacterial isolates. We found that 140 µM CAST inhibited α-glucosidase activity by 30% in a pure culture of Pseudomonas stutzeri. The α-glucosidase activity in Chryseobacterium gleum was inhibited by 90% at a concentration of 150 µM CAST, whereas the α-glucosidase in Paracoccus denitrificans was resistant to the inhibitor. CAST (140 µM) reduced α-glucosidase activity in activated sludge by 40%, the respiration rate being reduced by only 12%. No significant inhibition of the respiration rate was observed in Ps. stutzeri or Pa. denitrificans, whereas the respiration rate in C. gleum grown in a medium containing starch was inhibited by 50% with 140 µM CAST. No effect of CAST was observed in C. gleum grown in a complex medium. This indicated that CAST, at the concentration used, did not cause a general negative effect on bacterial activity. The results suggest that the CAST assay may potentially be useful in determining whether α-glucosidase activity, starch, poly- and disaccharides contribute appreciably to the overall activity of a bacterial community. However, the assay should not be used for quantitative measurements of such activity.
2-Amino-3-carboxy-1,4-naphthoquinone affects the end-product profile of bifidobacteria through the mediated oxidation of NAD(P)H by S. Yamazaki; T. Kaneko; N. Taketomo; K. Kano; T. Ikeda (pp. 72-78).
Glucose metabolism of bifidobacteria in the presence of 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), a specific growth stimulator for bifidobacteria, and ferricyanide (Fe(CN)6 3–) as an extracellular electron acceptor was examined using resting cells of Bifidobacterium longum and Bifidobacterium breve. NAD(P)H in the cells is oxidized by ACNQ with the aid of diaphorase activity, and reduced ACNQ donates the electron to Fe(CN)6 3–. Exogenous oxidation of NADH by the ACNQ/Fe(CN)6 3– system suppresses the endogenous lactate dehydrogenase reaction competitively, which results in the remarkable generation of pyruvate and a decrease in lactate production. In addition, a decrease in acetate generation is also observed in the presence of ACNQ and Fe(CN)6 3–. This phenomenon could not be explained in terms of the fructose-6-phosphate phosphoketolase pathway, but suggests rather that glucose is partially metabolized via the hexose monophosphate pathway. This was verified by NADP+-induced reduction of Fe(CN)6 3– in cell-free extracts in the presence of ACNQ. Effects of the ACNQ/Fe(CN)6 3– system on anaerobically harvested cells were also examined. Stoichiometric analysis of the metabolites from the pyruvate-formate lyase pathway suggests that exogenous oxidation of NADH is an efficient method to produce ATP in this pathway.
Degradation of phenanthrene, methylphenanthrenes and dibenzothiophene by a Sphingomonas strain 2mpII by T. Nadalig; N. Raymond; Ni'matuzahroh; M. Gilewicz; H. Budzinski; J. Bertrand (pp. 79-85).
Strain Sphingomonas sp. (2MPII), isolated from marine sediment, was able to utilize phenanthrene (P) or 2-methylphenanthrene (2MP) as the sole carbon source. However, 9-methylphenanthrene (9MP) and dibenzothiophene (DBT) were weakly degraded. The degradation rates of 9MP and DBT increased in the presence of 2MP, whilst the degradation rate of 2MP increased in the presence of 9MP. However, the presence of DBT inhibited the degradation of 2MP. DBT sulfone, a DBT metabolite, was not assimilated by the bacteria and its presence also decreased the degradation rate of 2MP.
Simultaneous detection and removal of organomercurial compounds by using the genetic expression system of an organomercury lyase from the transposon TnMERI1 by M. Narita; T. Yamagata; H. Ishii; C.-C. Huang; G. Endo (pp. 86-90).
Using a newly identified organomercury lyase gene (merB3) expression system from TnMERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.
Monitoring the denitrification of wastewater containing high concentrations of nitrate with methanol in a sulfur-packed reactor by I. Kim; S. Oh; M. Bum; J. Lee; S. Lee (pp. 91-96).
Biological denitrification of high nitrate-containing wastewater was examined in a sulfur-packed column using a smaller amount of methanol than required stoichiometrically for heterotrophic denitrification. In the absence of methanol, the observed nitrate removal efficiency was only about 40%, and remained at 400 mg NO3 –-N/l, which was due to an alkalinity deficiency of the pH buffer and of CO2 as a carbon source. Complete denitrification was achieved by adding approximately 1.4 g methanol/g nitrate-nitrogen (NO3 –-N) to a sulfur-packed reactor. As the methanol concentration increased, the overall nitrate removal efficiency increased. As influent methanol concentrations increased from 285 to 570, 855, and 1,140 mg/l, the value of Δ mg alkalinity as CaCO3 consumed/Δ mg NO3 –-N removed increased from –1.94 to –0.84, 0.24, and 0.96, and Δ mg SO4 2– produced/Δ mg NO3 –-N removed decreased from 4.42 to 3.57, 2.58, and 1.26, respectively. These results imply the co-occurrence of simultaneous autotrophic and heterotrophic denitrification. Sulfur-utilizing autotrophic denitrification in the presence of a small amount of methanol is very effective at decreasing both sulfate production and alkalinity consumption. Most of methanol added was removed completely in the effluent. A small amount of nitrite accumulated in the mixotrophic column, which was less than 20 mg NO2 – -N/l , while under heterotrophic denitrification conditions, nitrite accumulated steadily and increased to 60 mg NO2 – -N/l with increasing column height.
A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli by L. Khatib; Y. Tsai; B. Olson (pp. 97-104).
This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2–4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >105 E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.
Assessment of effluent turbidity in mesophilic and thermophilic activated sludge reactors – origin of effluent colloidal material by J. Vogelaar; J. van Lier; B. Klapwijk; M. de Vries; G. Lettinga (pp. 105-111).
Two lab-scale plug flow activated sludge reactors were run in parallel for 4 months at 30 and 55°C. Research focussed on: (1) COD (chemical oxygen demand) removal, (2) effluent turbidity at both temperatures, (3) the origin of effluent colloidal material and (4) the possible role of protozoa on turbidity levels. Total COD removal percentages over the whole experimental period were 66±7% at 30°C and 53±11% at 55°C. Differences in total COD removal between both systems were due to less removal of soluble and colloidal COD at 55°C compared to the reference system. Thermophilic effluent turbidity was caused by a combination of influent colloidal particles that were not effectively retained in the sludge flocs, and erosion of the thermophilic activated sludge itself, as shown by denaturing gradient gel electrophoresis (DGGE) profiles. DGGE analysis of PCR-amplified 16S rDNA fragments from mesophilic and thermophilic sludge differed, indicating that different microbial communities were present in the two reactor systems. The effects of protozoal grazing on the effluent turbidity of both reactors was negligible and thus could not account for the large turbidity differences observed.
Aerobic degradation of 2,4,6-trichlorophenol by a microbial consortium – selection and characterization of microbial consortium by L. Kharoune; M. Kharoune; J. Lebeault (pp. 112-117).
A microbial consortium that efficiently degrades 2,4,6-TCP (2,4,6-trichlorophenol), as the sole source of carbon and energy under aerobic conditions was selected from municipal activated sludge. Six bacterial strains, designated S1, S2, S3, S4, S5 and S6, were isolated from the selected consortium and five were identified as Sphingomonas paucimobilis (S2, S3), Burkholderia cepacia (S4), Chryseomonas luteola (S5) and Vibrio metschnikovii (S6). After prolonged cultivation followed by successive transfers, the consortium's degradation ability was improved and reached a specific degradation rate of 34 mg 2,4,6-TCP g–1 dry weight h–1 (about 51 mg 2,4,6-TCP g–1 cell protein h–1). The soluble chemical oxygen demand, chloride and oxygen uptake balance data clearly indicate the complete dechlorination and mineralization of 2,4,6-TCP. The consortium's activity was not inhibited by 2,4,6-TCP concentrations ≤400 mg l–1. The highest specific growth rate (µ max of 0.01 h–1) was achieved at 2,4,6-TCP concentration ≤200 mg l–1, and the inhibition constant (K i) was 610 mg l–1 according to the Haldane inhibition kinetic model. An inoculum size up to 120 mg l–1 cell dry weight is desirable in order to avoid a lag phase and to allow faster degradation of 2,4,6-TCP.
Developing and sustaining 3-chlorophenol-degrading populations in up-flow anaerobic column reactors under circum-denitrifying conditions by H.S. Bae; T. Yamagishi; Y. Suwa (pp. 118-124).
Microbial consortia capable of degrading 3-chlorophenol (3-CP) were enriched in continuous up-flow column reactors under circum-denitrifying conditions. 3-CP degradation capability was developed and sustained when 3-CP was supplied at 16–21 µM, although suppression of the 3-CP degradation capability was observed when 3-CP was supplied at 42 µM. When 3-CP was stably degraded, the ratio of nitrate consumption to 3-CP degradation approached the theoretical stoichiometric value, which was calculated by assuming a 3-CP degradation-dependent nitrate reduction. Batch-incubation experiments demonstrated that the microbial consortium that was enriched in the column reactors required either nitrate or oxygen for degrading 3-CP, while 3-CP was not degraded under sulfate-degrading conditions. Although many attempts were made to sustain the microbial 3-CP degradation capability under denitrifying conditions, mostly in batch cultures, none of them have been successful so far. Therefore, the results obtained in this study may be the first to demonstrate sustainable 3-CP degradation capability under circum-denitrifying conditions.