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Applied Microbiology and Biotechnology (v.62, #2-3)

The Corynebacterium glutamicum genome: features and impacts on biotechnological processes by M. Ikeda; S. Nakagawa (pp. 99-109).

Corynebacterium glutamicum has played a principal role in the progress of the amino acid fermentation industry. The complete genome sequence of the representative wild-type strain of C. glutamicum, ATCC 13032, has been determined and analyzed to improve our understanding of the molecular biology and physiology of this organism, and to advance the development of more efficient production strains. Genome annotation has helped in elucidation of the gene repertoire defining a desired pathway, which is accelerating pathway engineering. Post genome technologies such as DNA arrays and proteomics are currently undergoing rapid development in C. glutamicum. Such progress has already exposed new regulatory networks and functions that had so far been unidentified in this microbe. The next goal of these studies is to integrate the fruits of genomics into strain development technology. A novel methodology that merges genomics with classical strain improvement has been developed and applied for the reconstruction of classically derived production strains. How can traditional fermentation benefit from the C. glutamicum genomic data? The path from genomics to biotechnological processes is presented.

Evolution of catabolic pathways for synthetic compounds: bacterial pathways for degradation of 2,4-dinitrotoluene and nitrobenzene by G. R. Johnson; J. C. Spain (pp. 110-123).

The pathways for 2,4-dinitrotoluene (2,4-DNT) and nitrobenzene offer fine illustrations of how the ability to assimilate new carbon sources evolves in bacteria. Studies of the degradation pathways provide insight about two principal strategies for overcoming the metabolic block imposed by nitro- substituents on aromatic compounds. The 2,4-DNT pathway uses novel oxygenases for oxidative denitration and subsequent ring-fission. The nitrobenzene pathway links facile reduction of the nitro- substituent, a novel mutase enzyme, and a conserved operon encoding aminophenol degradation for mineralization of nitrobenzene. Molecular genetic analysis with comparative biochemistry reveals how the pathways were assembled in response to the recent appearance of the two synthetic chemicals in the biosphere.

Pilot plant for bioremediation of mercury-containing industrial wastewater by I. Wagner-Döbler (pp. 124-133).

Mercury is an extremely toxic pollutant that is currently being emitted mainly by low level industrial sources. It is distributed globally through the atmosphere, from where it precipitates onto the surface of the Earth, enters aquatic organisms, accumulates in fish and finally affects the health of human populations. Microbes have evolved a mechanism for mercury detoxification [mercury resistance operon (mer)] based on intracellular reduction of Hg²⁺ to non-toxic Hg⁰ by the mercuric reductase enzyme and subsequent diffusional loss of Hg⁰ from the cell. It was shown that Hg⁰ produced by microbial detoxification can be retained quantitatively in packed bed bioreactors, in which biofilms of mercury-resistant bacteria are grown on porous carrier material. This review describes operation of this system on a technical, fully automated, scale, and its operation at a chloralkali electrolysis factory. It was shown to work with high efficiency under fluctuating mercury concentrations and to be robust against transiently toxic conditions. The gradient of mercury concentration in the technical scale system exerted a strong selective pressure on the microbial community, which resulted in a succession of mercury-resistant strains at high mercury concentrations and an increase in phylogenetic and functional diversity at low mercury concentrations. Clean-up of mercury-containing wastewater by mercury-resistant microbes is a simple, environmentally friendly and cost-effective alternative to current treatment technologies.

Microbial iron respiration: impacts on corrosion processes by A. K. Lee; D. K. Newman (pp. 134-139).

In this review, we focus on how biofilms comprising iron-respiring bacteria influence steel corrosion. Specifically, we discuss how biofilm growth can affect the chemistry of the environment around the steel at different stages of biofilm development, under static or dynamic fluid regimes. We suggest that a mechanistic understanding of the role of biofilm metabolic activity may facilitate corrosion control.

Bioflavoring and beer refermentation by B. Vanderhaegen; H. Neven; S. Coghe; K. J. Verstrepen; G. Derdelinckx; H. Verachtert (pp. 140-150).

Various techniques are used to adjust the flavors of foods and beverages to new market demands. Although synthetic flavoring chemicals are still widely used, flavors produced by biological methods (bioflavors) are now more and more requested by consumers, increasingly concerned with health and environmental problems caused by synthetic chemicals. Bioflavors can be extracted from plants or produced with plant cell cultures, microorganisms or isolated enzymes. This Mini-Review paper gives an overview of different systems for the microbial production of natural flavors, either de novo, or starting with selected flavor precursor molecules. Emphasis is put on the bioflavoring of beer and the possibilities offered by beer refermentation processes. The use of flavor precursors in combination with non-conventional or genetically modified yeasts for the production of new products is discussed.

Enhancement of taxol production and release in Taxus chinensis cell cultures by ultrasound, methyl jasmonate and in situ solvent extraction by J. Wu; L. Lin (pp. 151-155).

This study evaluates the use of a novel mechanical stimulus, ultrasound (US), and a putative chemical elicitor, methyl jasmonate (MJ), combined with in situ solvent extraction (two-phase culture), to enhance taxol production by Taxus chinensis cells in suspension culture. The volumetric taxol yield was increased 1.5- to 1.8-fold with 2 min US treatment once or twice during a 4-week culture period, about 5-fold with 60–120 μM MJ, and 7- to 9-fold by in situ solvent extraction of taxol with dibutyl phthalate (DBP) (11% v/v). The percent of extracellular taxol or taxol release was also significantly increased. The combined use of US (day 5 or 9) and MJ treatment (day 7) resulted in taxol yields 20–50% higher than each of the treatments used alone. The most favorable strategy for taxol production was the application of US or MJ treatment, followed by in situ solvent extraction, giving rise to a taxol yield of 33–35 mg/l, about 17-fold higher than the control, at 1.9 mg/l. It was found that the organic solvent DBP, as well as US and MJ, stimulated the enzyme activity of secondary metabolic pathways, which was partially responsible for the enhanced taxol production.

Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer-controlled feeding strategy by M. J. Bailey; J. Tähtiharju (pp. 156-162).

A low-foaming hydrophobin II deletant of the Trichoderma reesei strain Rut-C30 was used for production of cellulases by continuous cultivation on lactose medium in a laboratory fermenter. The control paradigm of the addition of new medium to the continuous process was based on the growth dynamics of the fungus. A decrease in the rate of base addition to the cultivation for pH-minimum control was used as an indicator of imminent exhaustion of carbon source for growth and enzyme induction. When the amount of base added per 5 min computation cycle decreased below a given value, new medium was added to the fermenter. When base addition for pH control thereafter increased above the criterion value, due to increased growth, the medium feed was discontinued or decreased. The medium feeding protocol employed was successful in locking the fungus in the stage of imminent, but not actual, exhaustion of carbon source. According to the results of a batch cultivation of the same strain on the same medium, this is the phase of maximal enzyme productivity. The medium addition protocol used in this work resulted in a very stable continuous process, in which cellulase productivity was maintained for several hundred hours at the maximum level observed in a batch cultivation for only about 10 h. Despite a major technical disturbance after about 420 h, the process was restored to stability. When the cultivation was terminated after 650 h, the level of enzyme production was still maximal, with no signs of instability of the process.

Influence of growth conditions on bacteriocin production by Brevibacterium linens by A. S. Motta; A. Brandelli (pp. 163-167).

The influence of temperature, NaCl concentration and cheese whey media on growth of Brevibacterium linens ATCC 9175 and production of bacteriocin-like antimicrobial activity was studied. Bacteriocin production and activity were higher at 25°C than at 30°C. No significant growth or production of bacteriocins was observed at 37°C. When bacteriocin production was investigated in media containing different concentrations of NaCl, increased activity was observed in media containing 40 or 80 g l⁻¹, but not 120 g l⁻¹ NaCl. The addition of NaCl resulted in a significant increase in specific production rates of bacteriocin-like activity. Antimicrobial activity was also observed by cultivation of B. linens at 25°C in cheese whey media.

Influence of residual ethanol concentration on the growth of Gluconacetobacter xylinus I 2281 by H. Kornmann; P. Duboc; P. Niederberger; I. Marison; U. von Stockar (pp. 168-173).

The influence of residual ethanol on metabolism of food grade Gluconacetobacter xylinus I 2281 was investigated during controlled cultivations on 35 g/l glucose and 5 g/l ethanol. Bacterial growth was strongly reduced in the presence of ethanol, which is unusual for acetic acid bacteria. Biomass accumulated only after complete oxidation of ethanol to acetate and carbon dioxide. In contrast, bacterial growth initiated without delay on 35 g/l glucose and 5 g/l acetate. It was found that acetyl CoA was activated by the acetyl coenzyme A synthetase (Acs) pathway in parallel with the phosphotransacetylase (Pta)-acetate kinase (Ack) pathway. The presence of ethanol in the culture medium strongly reduced Pta activity while Acs and Ack remained active. A carbon balance calculation showed that the overall catabolism could be divided into two independent parts: upper glycolysis linked to glucose catabolism and lower glycolysis liked to ethanol catabolism. This calculation showed that the carbon flux through the tricarboxylic cycle is lower on ethanol than on acetate. This corroborated the diminution of carbon flux through the Pta-Ack pathway due to the inhibition of Pta activity on ethanol.

Multi parameter in vitro testing of ratjadone using flow cytometry by A. Burzlaff; M. Kalesse; C. Kasper; T. Scheper (pp. 174-179).

Ratjadone, isolated from the myxobacterium Sorangium cellulosum, belongs to the family of so-called orphan ligands, which includes leptomycin, callystatin and other compounds. In previous screening tests, ratjadone revealed a growth inhibitory effect against bacteria, yeast and human cancer cells. Following these first results, ratjadone was tested on several human tumour cell lines (Jurkat, HepG2, U87-MG) and, as a control, on a non-tumour cell line (RLC18) for its mode of action. The cell analysis was carried out by flow cytometry. This comprised cell density measurements, live-dead analysis, cell-cycle analysis and detection of apoptosis. First experiments confirmed the growth inhibitory effect on any chosen tumour cell line. Following these results a dose effect relationship was monitored, confirming the high effectiveness of ratjadone against cell growth at nanomolar concentration. Cell cycle analysis has shown that ratjadone intervenes in the cell cycle by arresting the cells in G1-phase. Biological testing of additional ratjadone derivatives with changed configuration and stereochemistry, identified the pharmacophoric site of the molecule.

Gene cloning, expression, and crystallization of a thermostable exo-inulinase from Geobacillus stearothermophilus KP1289 by Y. Tsujimoto; A. Watanabe; K. Nakano; K. Watanabe; H. Matsui; K. Tsuji; T. Tsukihara; Y. Suzuki (pp. 180-185).

The gene (inuA) encoding exo-inulinase (EC 3.2.1.80) was cloned from the thermophilic Geobacillus stearothermophilus (Bacillus stearothermophilus) KP 1289 growing at between 41°C and 69°C. The inuA gene consisted of 1,482 bp encoding a protein of 493 amino acids. The deduced polypeptide of molecular mass (M) 56,744 Da showed strong sequence similarity to Pseudomonas mucidolens exo-inulinase, Bacillus subtilis levanase, Paenibacillus polymyxa (Bacillus polymyxa) fructosyltransferase, and so on, indicating that the enzyme belonged to glycosyl hydrolase family 32. The M of the purified exo-inulinase, expressed in Escherichia coli HB101, was estimated as approximately 54,000 Da by both SDS-PAGE and gel filtration. These results suggested that the active form of the enzyme is a monomer. The enzyme was active between 30 and 75°C with an optimum at 60°C. The properties were identical to those of the native enzyme. Additionally, for the first time for a prokaryotic GH32 protein, crystals of the recombinant enzyme were obtained.

Cloning and characterization of the gene coding for the aerobic azoreductase from Pigmentiphaga kullae K24 by S. Blümel; A. Stolz (pp. 186-190).

The gene coding for an aerobic azoreductase was cloned from Pigmentiphaga kullae K24, which is able to grow with the carboxylated azo compound 1-(4′-carboxyphenylazo)-4-naphthol (carboxy-Orange I) as sole source of carbon and energy. The gene encoded a protein with a molecular weight of 20,557 Da, with a conserved putative NAD(P)H-binding site in the amino-terminal region. The deduced amino acid sequence showed no further significant sequence homologies to previously studied aerobic azoreductases. The azoreductase was heterologously expressed in Escherichia coli and shown to convert the sulfonated azo dye Orange I and furthermore Magneson II [4-(4-nitrophenylazo)-1-naphthol].

Molecular, biochemical and ecological characterisation of a bio-catalytic calcification reactor by F. Hammes; N. Boon; G. Clement; J. de Villiers; S. D. Siciliano; W. Verstraete (pp. 191-201).

Bio-catalytic calcification (BCC) reactors utilise microbial urea hydrolysis by autochthonous bacteria for the precipitation-removal of calcium, as calcite, from industrial wastewater. Due to the limited knowledge available concerning natural ureolytic microbial calcium carbonate (CaCO₃) precipitation, the microbial ecology of BCC reactors has remained a black box to date. This paper characterises BCC reactor evolution from initialisation to optimisation over a 6-week period. Three key parameters were studied: (1) microbial evolution, (2) the (bio)chemical CaCO₃ precipitation pathway, and (3) crystal nucleation site development. Six weeks were required to establish optimal reactor performance, which coincided with an increase in urease activity from an initial 7 mg urea l⁻¹ reactor h⁻¹ to about 100 mg urea l⁻¹ reactor h⁻¹. Urease activity in the optimal period was directly proportional to Ca²⁺ removal, but urease gene diversity was seemingly limited to a single gene. Denaturing gradient gel electrophoresis of 16S rRNA genes revealed the dynamic evolution of the microbial community structure of the calcareous sludge, which was eventually dominated by a few species including Porphyromonas sp., Arcobacter sp. and Bacteroides sp. Epi-fluorescence and scanning electron microscopy showed that the calcareous sludge was colonised with living bacteria, as well as the calcified remains of organisms. It appears that the precipitation event is localised in a micro-environment, due to colonisation of crystal nucleation sites (calcareous sludge) by the precipitating organisms.

cDNA cloning and functional expression of α-glucosidase from Mortierella alliacea by Y. Tanaka; T. Aki; K. Ishihara; S. Kawamoto; S. Shigeta; K. Ono (pp. 202-209).

We recently purified an α-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant α-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the α-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure–function relationship of the α-glucosidase with unique substrate specificity.

Cloning and characterisation of a glucoamylase gene (GlaM) from the dimorphic zygomycete Mucor circinelloides by J. Houghton-Larsen; P. A. Pedersen (pp. 210-217).

This article reports a novel strategy for the cloning of glucoamylase genes using conserved sequences and semi-nested PCR and its application in cloning the GlaM glucoamylase gene and cDNA from the dimorphic zygomycete Mucor circinelloides. The deduced 609-amino-acid enzyme (including signal peptide) is 63% identical to the Rhizopus oryzae raw starch-degrading glucoamylase and is the third glucoamylase reported to have the putative starch-binding domain placed N-terminally. The C-terminal catalytic domain is separated from the starch-binding domain by a serine/threonine-rich linker. An alignment of the cloned gene and cDNA sequences showed that the gene contains three introns. The transcriptional start site and the site of polyadenylation were defined by primer extension and 3'RACE, respectively. The atypical Kozak sequence is identical to the one used in R. oryzae in positions −1 to −4. Northern slot blots revealed that glucoamylase transcription is induced during growth on starch and repressed by glucose. In silico analysis of the 1.9-kb promoter sequence cloned by inverse PCR revealed the presence of several putative regulatory elements, most notably a 19-bp sequence containing six overlapping copies of the Saccharomyces cerevisiae Nrg1p binding sequence.

Development of a biosensor for on-line detection of tributyltin with a recombinant bioluminescent Escherichia coli strain by G. Thouand; H. Horry; M. J. Durand; P. Picart; L. Bendriaa; P. Daniel; M. S. DuBow (pp. 218-225).

A biosensor was developed for the detection of tributyltin (TBT), using a bioluminescent recombinant Escherichia coli::luxAB strain. Dedicated devices allowed the on-line measurement of bioluminescence, pH and dissolved oxygen values and the feed-back regulation of temperature. Bacterial physiology was monitored by the measurement of the cellular density, respiratory activity and the intracellular level of ATP, glucose and acetate levels. Our results showed that a synthetic glucose medium gave a better TBT detection limit than LB medium (respectively 0.02 µM and 1.5 µM TBT). High growth and dilution rates (D=0.9 h⁻¹) allowed maximum light emission from the bacterium. Moreover, simple atmospheric air bubbling was sufficient to provide oxygen for growth and the bioluminescence reaction. Real-time monitoring of bioluminescence after TBT induction occurred with continuous addition of decanal up to 300 µM, which was not toxic throughout a 7-day experiment. The design of our biosensor and the optimization of the main parameters that influence microbial activity led to the capacity for the detection of TBT.

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface by Y. Lin; T. Tsumuraya; T. Wakabayashi; S. Shiraga; I. Fujii; A. Kondo; M. Ueda (pp. 226-232).

A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of α-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.

Antagonistic properties of two recombinant strains of Streptomyces melanosporofaciens obtained by intraspecific protoplast fusion by S. Agbessi; J. Beauséjour; C. Déry; C. Beaulieu (pp. 233-238).

Intraspecific protoplast fusion was used to produce stable prototrophic recombinants of Streptomyces melanosporofaciens EF-76, a biocontrol agent of plant disease producing geldanamycin. Two recombinant strains (FP-54 and FP-60) that differed with regard to their antagonistic properties against Bacillus cereus ATCC 14579, Streptomyces scabies EF-35 and Phytophthora fragariae var. rubi 390 were characterized. FP-60 lost the ability to inhibit the in vitro growth of these microbial strains while FP-54 exhibited higher antagonistic activities against them. FP-60 was deficient in geldanamycin biosynthesis whereas FP-54 was shown to produce, in addition to geldanamycin, at least two other antimicrobial compounds that were absent in the culture supernatants of strain EF-76. Like the wild-type strain EF-76, strain FP-54 reduced common scab symptoms on potato tuber but no significant difference was observed between the disease index attributed to tubers treated with strain EF-76 or with strain FP-54. Strain FP-60 showed no protective effect against common scab. The disease index of tubers treated with this recombinant was worse than the index associated with potato tubers from control treatments.

Effect of cysteine desulfhydrase gene disruption on l-cysteine overproduction in Escherichia coli by N. Awano; M. Wada; A. Kohdoh; T. Oikawa; H. Takagi; S. Nakamori (pp. 239-243).

In Escherichia coli, the enzyme called cysteine desulfhydrase (CD), which is responsible for l-cysteine degradation, was investigated by native-PAGE and CD activity staining of crude cell extracts. Analyses with gene-disrupted mutants showed that CD activity resulted from two enzymes: tryptophanase (TNase) encoded by tnaA and cystathionine β-lyase (CBL) encoded by metC. It was also found that TNase synthesis was induced by the presence of l-cysteine. The tnaA and metC mutants transformed with the plasmid containing the gene for feedback-insensitive serine acetyltransferase exhibited higher l-cysteine productivity than the wild-type strain carrying the same plasmid. These results indicated that TNase and CBL did act on l-cysteine degradation in E. coli cells.

Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production by Md. M. Kabir; K. Shimizu (pp. 244-255).

For the efficient production of poly(3-hydroxybutyrate) (PHB) using recombinant Escherichia coli, it is of primal importance to overproduce NADPH, which is necessary for the PHB synthetic pathway. In order to overproduce NADPH in the pentose phosphate (PP) pathway, a recombinant E. coli was constructed in which the phosphoglucose isomerase (pgi) gene was knocked out to force the carbon flow into the PP pathway. The fermentation characteristics of the recombinant E. coli mutant lacking pgi were then investigated to determine the effect of overproduction of NADPH on efficient PHB production. It was found that, compared with the parent strain (E. coli JM109), growth of the E. coli mutant lacking pgi (E. coli DF11) is repressed due to NADPH overproduction in the PP pathway. Furthermore, repressed cell growth can be recovered to some extent by introducing a NADPH-consuming pathway, such as the PHB synthetic pathway. Efficient PHB production using such recombinant E. coli (DF11/pAeKG1) could be attained by appropriately controlling the glucose concentration in the fermentor. Total gene expression was investigated at the protein level by two-dimensional electrophoresis. Out of 22 differentially expressed proteins, 12 were identified with the aid of MALDI-TOF mass spectrometry. Variations in the accumulation of PHB in the recombinant pgi mutant carrying phb (E. coli DF11/pAeKG1) corresponded to the expression of proteins encoded by rpsA, znuA, fabD, potD, fkpA, gapA, ynaF and ibpA. The unfavorable conditions generated by PHB accumulation in the pgi mutant carrying phb resulted in the highest expression of 30S ribosomal protein S1, which ultimately caused a further increase in soluble protein synthesis.

Roles of tert-butyl formate, tert-butyl alcohol and acetone in the regulation of methyl tert-butyl ether degradation by Mycobacterium austroafricanum IFP 2012 by A. François; L. Garnier; H. Mathis; F. Fayolle; F. Monot (pp. 256-262).

Mycobacterium austroafricanum IFP 2012 is a Gram-positive strain able to grow on methyl tert-butyl ether (MTBE) as a sole carbon and energy source. The effect of two downstream metabolites of MTBE, tert-butyl formate (TBF) and tert-butyl alcohol (TBA) on MTBE degradation was investigated using resting cells. The addition of low concentrations of TBF decreased the MTBE degradation rate by about 30%. In contrast, the addition of TBA did not have a significant effect on MTBE degradation rate, even at high concentrations; and it was also shown that TBA degradation occurred only once MTBE was exhausted. At neutral pH, TBF hydrolysis involved mainly an esterase-type activity regulated by the presence of TBA. The TBF degradation rate was about four times lower than the MTBE degradation rate. Furthermore, acetone was identified as an intermediate during TBA degradation. An acetone mono-oxygenase activity, inhibited by methimazole but not by acetylene, was suggested. It was different from the MTBE/TBA mono-oxygenase and, thus, acetone did not appear to compete with MTBE and TBA for the same enzyme. These new results show that the metabolic regulation of the early steps of MTBE degradation by M. austroafricanum IFP 2012 is complex, involving inhibition and competition phenomena.

Novel proline hydroxylase activities in the pneumocandin-producing fungus Glarea lozoyensis responsible for the formation of trans 3- and trans 4-hydroxyproline by L. Petersen; R. Olewinski; P. Salmon; N. Connors (pp. 263-267).

Novel proline 3-hydroxylase (P3H) and proline 4-hydroxylase (P4H) activities that convert free l-proline to both trans 3- and trans 4-hydroxy-l-proline were detected in protein extracts of the anamorphic fungus Glarea lozoyensis. The enzymatic conversion of l-proline to trans 3- and trans 4-hydroxy-l-proline was strictly dependent on α-ketoglutarate, ascorbate, and dithiothreitol. Ferrous iron was required for optimal P3H and P4H activity. These substrate and co-factor requirements indicate these enzyme activities belong to the class of 2-oxoglutarate-dependent dioxygenases. Both P3H and P4H were inhibited by zinc and other trace metals. The addition of proline to the fermentation medium resulted in an increase in the specific activity of P4H and a decrease in the synthesis of pneumocandin C₀. Additionally, the synthesis of trans 3- and trans 4-hydroxy-l-proline in vivo was affected differently by the proline concentration in the medium. This result suggested that two enzymes may be responsible for the regio- and stereospecific hydroxylation of l-proline.

Quantitative detection of crystalline lysine supplementation in poultry feeds using a rapid bacterial bioluminescence assay by I. B. Zabala Díaz; S. C. Ricke (pp. 268-273).

Lysine is an essential amino acid for both humans and animals; and it is usually the first or second limiting amino acid in most formulated diets. In order to estimate the lysine content in feeds and feed sources, rapid amino acid bioassays have been developed. The objective of this work is to assess a rapid assay for lysine supplementation in chicken feeds, using a luminescent Escherichia coli lysine-auxotrophic strain, to avoid prior thermal sterilization. An E. coli lysine auxotroph carrying a plasmid with lux genes was used as the test organism. The lysine assay was conducted using depleted auxotrophic cells in lysine samples. Luminescence was measured with a Dynex MLX luminometer after addition of the aldehyde substrate. Growth response (monitored as optical density at 600 nm) and light emission response of the assay E. coli strain were monitored to generate standard curves. Bioluminescent analysis of feed samples indicated that the method works well in the presence of a complex feed matrix. Comparison of both optical density and luminescent-based methods indicated that, when the assay takes place under optimal conditions, both methodologies correlated well (r ²=0.99). Except for the 0.64% lysine-supplemented feed, estimates for lysine based on the bacterial assay were over 80% (82–97%) of the theoretical values. Animal data showed that the bacterial bioluminescent method correlated well with the chick bioassay when diets with different levels of lysine supplementation were assayed for lysine bioavailability (r ²=0.97). Luminescent methodology coupled with a bacterial growth assay is a promising technique to assess lysine availability in supplemented animal feeds.

Removal of inorganic and organic mercurials by immobilized bacteria having mer-ppk fusion plasmids by M. Kiyono; H. Omura; T. Omura; S. Murata; H. Pan-Hou (pp. 274-278).

Feasibility of biological mercury removal from wastewater was examined by using alginate-immobilized cells of Escherichia coli carrying mer-ppk fusion plasmid pMKB18. Immobilized cells engineered to express mercury-transport system, organomercurial lyase and polyphosphate efficiently removed organic and inorganic mercury from contaminated wastewater over a wide concentration range of mercurials, probably via intracellular accumulation mediated by ppk-specified polyphosphate. Bioaccumulation of mercury was selective compared to other metals such as Cd²⁺, Pb²⁺ and Cr⁶⁺. The immobilized cells could be used repeatedly (at least three times) without large loss of mercury removal activity. From these results, it is concluded that the mer-ppk fusion plasmid and the immobilized cells are useful for simultaneous removal of organic and inorganic mercury from contaminated wastewater.

Degradation and detoxification of endosulfan isomers by a defined co-culture of two Bacillus strains by N. Awasthi; A. K. Singh; R. K. Jain; B. S. Khangarot; A. Kumar (pp. 279-283).

The degradation of α and β isomers of endosulfan by a two-member bacterial co-culture was studied. Results were similar whether the two isomers were present individually or together, as in technical endosulfan. The degradation of both isomers was accompanied by the formation of endosulfan diol and endosulfan lactone. Accumulation of the metabolite, endosulfan sulfate was, however, not observed during the reaction with either of the isomers. The microbial degradation of endosulfan isomers was also accompanied by a decrease in its toxicity to the test organism Tubifex tubifex Müller.

A novel catabolic activity of Pseudomonas veronii in biotransformation of pentachlorophenol by I.-H. Nam; Y.-S. Chang; H.-B. Hong; Y.-E. Lee (pp. 284-290).

Pseudomonas veronii PH-05, a bacterial strain capable of transforming pentachlorophenol (PCP) to a metabolic intermediate, was isolated by selective enrichment of soil samples from a timber storage yard. Strain PH-05 was shown to be able to grow using PCP as the sole source of carbon and energy. GC-MS analysis showed that the metabolic intermediate was tetrachlorocatechol, which inhibited the growth of this strain. The formation of tetrachlorocatechol during biotransformation was monitored, and its inhibitory effect on growth of strain PH-05 was analyzed at a range of concentrations. The catabolic activity of the isolated strain differs from that of other PCP-degrading bacteria, which metabolize PCP through a chlorinated hydroquinone intermediate.

Biodegradation of polycyclic aromatic hydrocarbons in a two-phase partitioning bioreactor in the presence of a bioavailable solvent by C. T. MacLeod; A. J. Daugulis (pp. 291-296).

Mycobacterium PYR-1 was used in a two-phase partitioning bioreactor (TPPB) to degrade low and high molecular weight polycyclic aromatic hydrocarbons. TPPBs are characterized by a cell-containing aqueous phase, and an immiscible and biocompatible organic phase that partitions toxic substrates to the cells based on their metabolic demand and the thermodynamic equilibrium of the system. A bioavailable solvent, that is, a solvent usable as a carbon source, was used as the organic layer. Although bioavailable solvents are traditionally deemed unsuitable for use in TPPBs, bis(ethylhexyl) sebacate had superior chemical properties to other solvents examined and was cost-effective. In this system, 1 g of phenanthrene and 1 g of pyrene were completely degraded within 4 days, at rates of 168 mg l⁻¹ day⁻¹ and 138 mg l⁻¹day⁻¹, respectively, based on a 3-l aqueous volume. This is the highest pyrene degradation rate reported in the literature to date. Significant degradation of naphthalene and anthracene was also obtained. This work demonstrates that bioavailable solvents can be successfully used in TPPB systems, and may change the protocols commonly used to select solvents for TPPBs in the future.

The treatment of gaseous benzene by two-phase partitioning bioreactors: a high performance alternative to the use of biofilters by C. T. Davidson; A. J. Daugulis (pp. 297-301).

A 2-l (1-l working volume) two-phase partitioning bioreactor (TPPB) was used as an integrated scrubber/bioreactor in which the removal and destruction of benzene from a gas stream was achieved by the reactor's organic/aqueous liquid contents. The organic solvent used to trap benzene was n-hexadecane, and degradation of benzene was achieved in the aqueous phase using the bacterium Alcaligenes xylosoxidans Y234. A gas stream with a benzene concentration of 340 mg l⁻¹ at a flow rate of 0.414 l h⁻¹ was delivered to the system at a loading capacity of 140 g m⁻³ h⁻¹, and an elimination capacity of 133 g m⁻³h⁻¹ was achieved (the volume in this term is the total liquid volume of the TPPB). This elimination capacity is between 3 and 13 times greater than any benzene elimination achieved by biofiltration, a competing biological air treatment strategy. It was also determined that the evaluation of TPPB performance in terms of elimination capacity should include the cell mass present in the system, as this is a readily controllable quantity. A specific benzene utilization rate of 0.57 g benzene (g cells)⁻¹ h⁻¹ was experimentally determined in a bioreactor with a cell concentration that varied dynamically between 0.2 and 1 g l⁻¹. If it assumed that this specific benzene utilization rate (0.57 g g⁻¹ h⁻¹) is independent of cell concentration, then a TPPB operated at high cell concentrations could potentially achieve elimination capacities several hundred times greater than those obtained with biofilters.

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Applied Microbiology and Biotechnology (v.62, #2-3)

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Applied Microbiology and Biotechnology (v.62, #2-3)