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Applied Microbiology and Biotechnology (v.56, #5-6)

The use of micro-organisms for L-ascorbic acid production: current status and future perspectives by R. Hancock; R. Viola (pp. 567-576).

L-Ascorbic acid (L-AA) has been industrially produced for around 60 years in a primarily chemical process utilising D-glucose (D-glc) as starting material. Current world production is estimated at approximately 80,000 tonnes per annum with a worldwide market in excess of U.S. $600 million. We present a brief overview of research geared to exploiting micro-organisms for the industrial production of vitamin C, with emphasis on recent approaches using genetically engineered bacterial strains. We also discuss the potential for direct production of L-AA exploiting novel biochemical pathways with particular reference to yeast fermentations. The potential advantages of these novel approaches over current chemical and biotechnological processes are outlined.

The potential of genetic engineering for improving brewing, wine-making and baking yeasts by S. Dequin (pp. 577-588).

The end of the twentieth century was marked by major advances in life technology, particularly in areas related to genetics and more recently genomics. Considerable progress was made in the development of genetically improved yeast strains for the wine, brewing and baking industries. In the last decade, recombinant DNA technology widened the possibilities for introducing new properties. The most remarkable advances, which are discussed in this Mini-Review, are improved process performance, off-flavor elimination, increased formation of by-products, improved hygienic properties or extension of substrate utilization. Although the introduction of this technology into traditional industries is currently limited by public perception, the number of potential applications of genetically modified industrial yeast is likely to increase in the coming years, as our knowledge derived from genomic analyses increases.

Mating-type genes for classical strain improvements of ascomycetes by S. Pöggeler (pp. 589-601).

The ability to mate fungi in the laboratory is a valuable tool for genetic analysis and for classical strain improvement. In ascomycetous fungi, mating typically occurs between morphologically identical partners that are distinguished by their mating type. In most cases, the single mating-type locus conferring mating behavior consists of dissimilar DNA sequences (idiomorphs) in the mating partners. All ascomycete mating-type idiomorphs encode proteins with confirmed or putative DNA-binding motifs. These proteins control, as master regulatory transcription factors, pathways of cell speciation and sexual morphogenesis. Mating-type organization of four of the six classes of ascomycetes has been studied at the molecular level over the past 20 years. This review gives a short overview of the structural organization of the mating-type loci of yeasts and filamentous ascomycetes. In addition, this review describes how the availability of mating-type sequences allows the investigation of key issues concerning genetic and phylogenetic analyses of fungal species.

Mating-type genes for basidiomycete strain improvement in mushroom farming by E. Kothe (pp. 602-612).

Mushroom production is dependent on the quality of the spawn used to inoculate the cultures. In order to produce high-quality spawn, breeding programs for strains resistant to certain diseases and able to form high-quality fruit bodies under standard growth conditions are necessary. The investigation of the molecular basis for mating provides access to the use of mating-type genes in order to facilitate breeding. For research purposes, two mushroom-forming homobasidiomycetes have been used due to their easy cultivation and sexual propagation on defined minimal media: Schizophyllum commune and Coprinus cinereus. The mating-type genes control formation of the dikaryon from two haploid strains. Only the dikaryon is fertile and able to form mushrooms under the right environmental conditions. These genes are now used in mating-type-assisted breeding programs for economically important mushrooms, especially the white button mushroom, Agaricus bisporus, and the oyster mushroom, Pleurotus ostreatus, aiming at high-yield and high-quality standard mushroom production. Most mushroom species posses two mating-type loci that control their breeding. The genes encoded in the A loci lead to the formation of transcription factors that belong to the class of homeodomain proteins. Active transcription factors are formed by heterodimerization of two proteins of different allelic specificities. In nature, this is only the case if two cells of different mating type have fused to combine the different proteins in one cytoplasm. While fusion in homobasidiomycetes is found irrespectively of mating type, exchange of nuclei between mating mycelia is dependent on the products of the B mating-type loci. The B genes form a pheromone and receptor system that enables the fungi to initiate nuclear migration. The molecular details of the two genetic systems controlling breeding in basidiomycetes are presented in this review.

A system of categorizing enzyme–cell wall associations in Agaricus bisporus, using operational criteria by J. Sassoon; H. Mooibroek (pp. 613-622).

Enzymes were investigated for their occurrence in the cell wall fraction (4,000 g sediment of the homogenate) of Agaricus bisporus sporocarps. Besides the markers malate dehydrogenase (MalDH), hexokinase (HK) and ATPase, the range of entities studied included γ-glutamyl transferase (γ-GT), mannitol dehydrogenase (MDH), phenoloxidase, chitin and β-1,3-glucan synthases (ChS, β-GS), chitinase, β-N-acetylhexosaminidase (HexNAc′ase) and β-glucanase. Using the extractability in dilute buffer, digitonin and NaCl at high ionic strength as the operational criteria, four categories (I–IV) of enzyme–wall associations could be discerned: category I encompasses enzymes which are artefactually present (i.e. contaminants); category II, enzymes that are hydrophobically bound (which may or may not be genuinely wall-associated), III includes enzymes that are ionically bound and IV, enzymes whose bonding to the wall is in all probability covalent. The same enzyme entity may have representatives in more than one category, e.g. ChS and β-GS (I, II, IV), phenolase (I, II, III, IV), β-glucanase, chitinase and HexNAc′ase (I, IV). It is thought that the categorization presented could be of general applicability in fungi as well as in higher plants to specify enzyme–wall associations in a straightforward, comparable manner, thus avoiding some of the ambiguous terms prevailing in the literature, such as "weakly", "strongly" or "tightly" wall bound. The results are discussed in more detail for several of the more economically important enzymes studied.

Mass production of entomopathogenic nematodes for plant protection by Ralf-Udo Ehlers (pp. 623-633).

Entomopathogenic nematodes of the genera Heterorhabditis and Steinernema are commercially used to control pest insects. They are symbiotically associated with bacteria of the genera Photorhabdus and Xenorhabdus, respectively, which are the major food source for the nematodes. The biology of the nematode–bacterium complex is described, a historical review of the development of in vitro cultivation techniques is given and the current use in agriculture is summarised. Cultures of the complex are pre-incubated with the symbiotic bacteria before the nematodes are inoculated. Whereas the inoculum preparation and preservation of bacterial stocks follow standard rules, nematodes need special treatment. Media development is mainly directed towards cost reduction, as the bacteria are able to metabolise a variety of protein sources to provide optimal conditions for nematode reproduction. The process technology is described, discussing the influence of bioreactor design and process parameters required to obtain high nematode yields. As two organisms are grown in one vessel and one of them is a multicellular organism, the population dynamics and symbiotic interactions need to be understood in order to improve process management. Major problems can originate from the delayed or slow development of the nematode inoculum and from phase variants of the symbiotic bacteria that have negative effects on nematode development and reproduction. Recent scientific progress has helped to understand the biological and technical parameters that influence the process, thus enabling transfer to an industrial scale. As a consequence, costs for nematode-based products could be significantly reduced.

The cellulosome and cellulose degradation by anaerobic bacteria by W. Schwarz (pp. 634-649).

Despite its simple chemical composition, cellulose exists in a number of crystalline and amorphous topologies. Its insolubility and heterogeneity makes native cellulose a recalcitrant substrate for enzymatic hydrolysis. Microorganisms meet this challenge with the aid of a multi-enzyme system. Aerobic bacteria produce numerous individual, extra-cellular enzymes with binding modules for different cellulose conformations. Specific enzymes act in synergy to elicit effective hydrolysis. In contrast, anaerobic bacteria possess a unique extracellular multi-enzyme complex, called cellulosome. Up to 11 different enzymes are aligned on the non-catalytic scaffolding protein and thus ensure a high local concentration, together with the correct ratio and order of the components. These multi-enzyme complexes attach both to the cell envelope and to the substrate, mediating the proximity of the cells to the cellulose. Binding to the scaffolding stimulates the activity of each individual component towards the crystalline substrate. The most complex and best investigated cellulosome is that of the thermophilic bacterium Clostridium thermocellum, but a scheme for the cellulosomes of the mesophilic clostridia and the ruminococci emerges. Many crucial details of cellulose hydrolysis are still to be uncovered. Yet, a mechanistic model for the action of enzyme complexes on the surface of insoluble substrates becomes apparent and the application of enzymatic hydrolysis of cellulosic biomass can now be addressed.

Biodegradation and bioremediation of hydrocarbons in extreme environments by R. Margesin; F. Schinner (pp. 650-663).

Many hydrocarbon-contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, high salt concentrations, or high pressure. Hydrocarbon-degrading microorganisms, adapted to grow and thrive in these environments, play an important role in the biological treatment of polluted extreme habitats. The biodegradation (transformation or mineralization) of a wide range of hydrocarbons, including aliphatic, aromatic, halogenated and nitrated compounds, has been shown to occur in various extreme habitats. The biodegradation of many components of petroleum hydrocarbons has been reported in a variety of terrestrial and marine cold ecosystems. Cold-adapted hydrocarbon degraders are also useful for wastewater treatment. The use of thermophiles for biodegradation of hydrocarbons with low water solubility is of interest, as solubility and thus bioavailability, are enhanced at elevated temperatures. Thermophiles, predominantly bacilli, possess a substantial potential for the degradation of environmental pollutants, including all major classes. Indigenous thermophilic hydrocarbon degraders are of special significance for the bioremediation of oil-polluted desert soil. Some studies have investigated composting as a bioremediation process. Hydrocarbon biodegradation in the presence of high salt concentrations is of interest for the bioremediation of oil-polluted salt marshes and industrial wastewaters, contaminated with aromatic hydrocarbons or with chlorinated hydrocarbons. Our knowledge of the biodegradation potential of acidophilic, alkaliphilic, or barophilic microorganisms is limited.

Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas by D. Solaiman; R. Ashby; T. Foglia (pp. 664-669).

Pseudomonas putida and P. oleovorans have been extensively studied for their production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). These bacteria are incapable of metabolizing triacylglycerols (TAGs). We have constructed recombinant P. putida and P. oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis. A recombinant plasmid, pCN51lip-1, carrying Pseudomonas lipase genes was used to electrotransform these organisms. The transformants expressed TAG-hydrolyzing activity as shown by a rhodamine B fluorescence plate assay. The genetically modified organisms grew in TAG-containing medium to a cell dry weight of 2–4 g/l. The recombinant P. putida produced mcl-PHA at a crude yield of 0.9–1.6 g/l with lard or coconut oil (Co) as substrate. While P. oleovorans transformant did not produce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield of 0.5 g/l. Compositional analysis by gas chromatography/mass spectrometry showed that β-hydroxyoctanoate (31–45 mol %) and β-hydroxydecanoate (28–35 mol %) were the dominant repeat units of the TAG-based PHA. The number-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82–170×10³ g/mol and 464–693×10³ g/mol, respectively. The recombinant approach can greatly increase the number of organisms that can be used to produce PHA from fat and oil substrates.

Effect of exogenous lysine on the expression of early cephamycin C biosynthetic genes and antibiotic production in Nocardia lactamdurans MA4213 by A. Leitão; F. Enguita; J. Martín; Santos J. Oliveira (pp. 670-675).

In β-lactam producing microorganisms, the first step in the biosynthesis of the β-lactam ring is the condensation of three amino acid precursors: α-aminoadipate, L-cysteine and D-valine. In Nocardia lactamdurans and other cephamycin-producing actinomycetes, α-aminoadipate is generated from L-lysine by two sequential enzymatic steps. The first step involves a lysine-6-aminotransferase activity (LAT), considered to be one of the rate-limiting steps for antibiotic biosynthesis. Here, we report the effect of exogenous lysine on antibiotic production by N. lactamdurans MA4213. Lysine-supplemented cultures showed higher titers of cephamycin C, an effect that was more significant at early fermentation times. The increase in cephamycin C production was not quantitatively correlated with specific LAT activity in lysine-supplemented cultures. Observation of a positive effect of lysine on cephamycin C production by N. lactamdurans was dependent on carbon source availability in the culture media. Supplementation of the culture media with exogenous lysine did not affect the mRNA levels of the early biosynthetic genes controlled by the bidirectional promoter. These results indicate that L-lysine is required not only for antibiotic biosynthesis, but particularly as carbon or nitrogen source.

Propionic acid production in an in situ cell retention bioreactor by V. Goswami; A. Srivastava (pp. 676-680).

Continuous fermentations were conducted with in situ cell retention using spin filters (pore size 5 µm and 10 µm) for propionic acid production by Propionibacterium acidipropionici. Continuous fermentation with a 5-µm pore size spin filter resulted in 50% cell retention. Propionic acid productivity was enhanced (0.9 g l^–1h^–1) by approximately four-fold compared to conventional batch fermentation (0.25 g l^–1h^–1). The in situ cell retention (5-µm pore size spin filter) bioreactor was operated continuously and smoothly for 8 days at a dilution rate of D=0.05 h^–1.

Spacer-mediated display of active lipase on the yeast cell surface by M. Washida; S. Takahashi; M. Ueda; A. Tanaka (pp. 681-686).

We have constructed a Saccharomyces cerevisiae strain displaying an active lipase on the cell surface by cell surface engineering. The gene encoding Rhizopus oryzae lipase (ROL) was fused with the genes encoding the pre-α-factor leader sequence and the C-terminal half of α-agglutinin including the glycosylphosphatidylinositol-anchor attachment signal. The constructed gene was overexpressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance lipase activity by preserving the conformation of the active site near the C-terminal portion. Localization of the expressed ROL on the cell surface was confirmed by immunofluorescence microscopy. The ROL displayed on the yeast cell wall exhibited activity toward soluble 2,3-dimercaptopropan-1-ol tributyl ester (BALB) and insoluble triolein. The insertion of linker peptides effected the activity towards BALB, thereby demonstrating that the optimal length of linker peptides was present. The activity towards triolein was higher in lipases with longer linker peptides. ROL displayed on the cell wall exhibited a comparable and/or higher activity towards triolein than the secreted form of the enzyme. This is the first report of an active lipase displayed on the cell surface. Furthermore, insertion of a linker peptide of the appropriate length as a spacer may be an improved method to effectively display enzymes, especially those having the active region at the C-terminal portion, on the cell surface.

Pressure measurement to evaluate ethanol or lactic acid production during glucose fermentation by yeast or heterofermentative bacteria in pure and mixed culture by M. Wick; J.-M. Lebeault (pp. 687-692).

A rapid and simple technique to follow CO₂ release during fermentation of glucose by heterofermentative bacteria or yeasts was used in order to evaluate ethanol and lactate production in pure and mixed cultures of yeast and bacteria. In pure cultures, good correlations were found between gas pressure variations (ΔP) and ethanol or lactate production by yeasts or heterofermentative bacteria, and ratios between ΔP and ethanol or lactate produced could be established. In mixed cultures, ratios between maximal ΔP and total amount of glucose consumed were determined. It was thus possible to evaluate the amount of glucose that was consumed by each strain and then deduce the bacterial lactate production. Good results were obtained for mixed cultures of yeast and homofermentative bacteria. This technique may be useful to evaluate the activity of strains in mixed cultures of yeast and lactic acid bacteria.

An improved procedure for characterization of spatial and temporal evolution of immobilized cells in gel membranes by W. Zhang; A. Berry; C. Franco (pp. 693-699).

An improved procedure that allows the simple and reproducible characterization of spatial and temporal distribution of immobilized biomass in gel membranes was developed. This procedure involves three main steps in the preparation of membrane samples, the use of a standard microtome to obtain membrane slices, and the measurement of cell concentration by spectrophotometry. The key improvement in this procedure is to prepare the membrane samples by clamping them between two glass plates and storing them in a –80°C freezer for a specified period of time depending on the membrane thickness. With this simple pre-treatment, the membrane samples were frozen in an ideal physical state to be cut into flat, consistent, slices using a commercial freezing sledge microtome, thus providing accurate and reproducible results. As a validation case study, a gel membrane bioreactor was constructed in which an alginate gel membrane with immobilized Lactobacillus rhamnosus cells was flanked by two well-mixed chambers with identical fermentation media. The improved procedure was employed to experimentally determine the intra-membrane cell distribution in the alginate membranes during fermentation. The experimental results showed a heterogeneous "U-shape" biomass distribution across the membrane, with the highest cell concentration at the membrane-solution interface. High reproducibility and accuracy were verified by a low average standard deviation (<5%) and a high biomass recovery ratio (>90%), respectively.

Thioglucosidase activity from Sphingobacterium sp. strain OTG1 by G. Meulenbeld; S. Hartmans (pp. 700-706).

Screening for novel thioglucoside hydrolase activity resulted in the isolation of Sphingobacterium sp. strain OTG1 from enrichment cultures containing octylthioglucoside (OTG). OTG was hydrolysed into octanethiol and glucose by cell free extracts. Besides thioglucoside hydrolysis, several other glucoside hydrolase activities were detected in the Sphingobacterium sp. strain OTG1 cell free extract. By adding β-glucosidase inhibitors it was possible to discriminate between these different activities. Ascorbic acid and D-gluconic acid lactone inhibited the hydrolysis of p-nitrophenyl β-glucoside, but did not affect octyl- and octylthioglucoside hydrolase activity. Besides OTG, various other thioglucosides were hydrolysed by the novel thioglucosidase, with almost the same activities regardless of the nature of the aglycone, including the myrosinase model substrate sinigrin (a glucosinolate). Sinigrin could also be used as a growth substrate by Sphingobacterium sp. strain OTG1, although at concentrations exceeding 0.15 mM degradation was not complete.

Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals by T. Irie; Y. Honda; T. Hirano; T. Sato; H. Enei; T. Watanabe; M. Kuwahara (pp. 707-709).

It was reported that Pleurotus ostreatus was transformed unstably using recombinant plasmids containing a hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans expression signals, and that the plasmids were maintained extrachromosomally in the transformants. Here we report a stable and integrative transformation of the fungus to hygromycin B resistance, using a recombinant hph fused with Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase expression signals. Restriction-enzyme-mediated integration (REMI) was also tried and increased the transformation efficiency about ten-fold.

Effect of transketolase modifications on carbon flow to the purine-nucleotide pathway in Corynebacterium ammoniagenes by N. Kamada; A. Yasuhara; Y. Takano; T. Nakano; M. Ikeda (pp. 710-717).

Transketolase, one of the enzymes in the nonoxidative branch of the pentose phosphate pathway, operates to shuttle ribose 5-phosphate and glycolytic intermediates together with transaldolase, and might be involved in the availability of ribose 5-phosphate, a precursor of nucleotide biosynthesis. The tkt and tal genes encoding transketolase and transaldolase, respectively, were cloned from the typical nucleotide- and nucleoside-producing organism Corynebacterium ammoniagenes by a PCR approach using oligonucleotide primers derived from conserved regions of each amino acid sequence from other organisms. Enzymatic and molecular analyses revealed that the two genes were clustered on the genome together with the glucose 6-phosphate dehydrogenase gene (zwf). The effect of transketolase modifications on the production of inosine and 5′-xanthylic acid was investigated in industrial strains of C. ammoniagenes. Multiple copies of plasmid-borne tkt caused about tenfold increases in transketolase activity and resulted in 10–20% decreased yields of products relative to the parents. In contrast, site-specific disruption of tkt enabled both producers to accumulate 10–30% more products concurrently with a complete loss of transketolase activity and the expected phenotype of shikimate auxotrophy. These results indicate that transketolase normally shunts ribose 5-phosphate back into glycolysis in these biosynthetic processes and interception of this shunt allows cells to redirect carbon flux through the oxidative pentose pathway from the intermediate towards the purine-nucleotide pathway.

Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems. by S. Guerrero; H.-J. Hecht; B. Hofmann; H. Biebl; M Singh (pp. 718-723).

The amino acid analogue selenomethionine (SeMet) is shown to be efficiently incorporated into recombinant proteins expressed in Escherichia coli grown in a simple minimal medium without the addition of synthetic amino acids. Furthermore, satisfactory SeMet incorporation is obtained with a methionine-prototrophic strain transformed with commonly used vector systems. As examples, purified tryparedoxin 1 from Crithidia fasciculata, alkylhydroperoxide reductase (AhpC) from Mycobacterium marinum and the 16-kDa antigen from M. tuberculosis are shown to be efficiently labelled with SeMet, using the culture conditions and the host/vector systems described here. Enzymatic analysis reveals no differences between native and SeMet-labelled tryparedoxin 1 enzyme. Both proteins yield crystals under similar conditions. The culture conditions and host vector systems described greatly facilitate selenium-labelling of proteins for 3-D structure determination.

Characterization of the eugenol hydroxylase genes (ehyA/ehyB) from the new eugenol-degrading Pseudomonas sp. strain OPS1 by K. Brandt; S. Thewes; J. Overhage; H. Priefert; A. Steinbüchel (pp. 724-730).

During the screening for bacteria capable of converting eugenol to vanillin, strain OPS1 was isolated, which was identified as a new Pseudomonas species by 16 s rDNA sequence analysis. When this bacterium was grown on eugenol, the intermediates, coniferyl alcohol, ferulic acid, vanillic acid, and protocatechuic acid, were identified in the culture supernatant. The genes encoding the eugenol hydroxylase (ehyA, ehyB), which catalyzes the first step of this biotransformation, were identified in a genomic library of Pseudomonas sp. strain OPS1 by complementation of the eugenol-negative mutant SK6165 of Pseudomonas sp. strain HR199. EhyA and EhyB exhibited 57% and 85% amino acid identity to the eugenol hydroxylase subunits of Pseudomonas sp. strain HR199 and up to 34% and 54% identity to the corresponding subunits of p-cresol methylhydroxylase from P. putida. Moreover, the amino-terminal sequences of the α- and β-subunits reported recently for an eugenol dehydrogenase of P. fluorescens E118 corresponded well with the appropriate regions of EhyA and EhyB. Downstream of ehyB, an open reading frame was identified, whose deduced amino acid sequence exhibited up to 71% identity to azurins, representing most probably the gene (azu) of the physiological electron acceptor of the eugenol hydroxylase. The eugenol hydroxylase genes were amplified by PCR, cloned, and functionally expressed in Escherichia coli.

Characterization of the lipA gene encoding the major lipase from Pseudomonas aeruginosa strain IGB83 by A. Martínez; G. Soberón-Chávez (pp. 731-735).

The lipases produced by Pseudomonas have a wide range of potential biotechnological applications. Pseudomonas aeruginosa IGB83 was isolated as a highly lipolytic strain which produced a thermotolerant and alkaline lipase. In the present work, we have characterized the P. aeruginosa IGB83 gene (lipA) encoding this enzyme. We describe the construction of a lipA mutant and report on the effect of two carbon sources on lipase expression.

The mer operon of a mercury-resistant Pseudoalteromonas haloplanktis strain isolated from Minamata Bay, Japan by K. Iohara; R. Iiyama; K. Nakamura; S. Silver; M. Sakai; M. Takeshita; K. Furukawa (pp. 736-741).

A mer operon of mercury-resistant Pseudoalteromonas haloplanktis strain M1, isolated from sea water of Minamata Bay, was cloned and analyzed. The mer genes were located in the chromosome and organized as merR-merT-merP-merC-merA-merD, the same order as that in Tn21. However, the orientation of the merR gene is the same as that of other mer genes (opposite direction to Tn21), and merR was cotranscribed with other mer genes, a pattern that has not been previously seen with mer determinants from other Gram-negative bacteria. Furthermore, the amino acid similarities of the corresponding mer gene products between those from strain M1 and Tn21 were unusually low.

Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay by T. Gotoh; Y. Miyazaki; K.-I. Kikuchi; W. Bentley (pp. 742-749).

Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-infected cells all the time and their level in the medium continuously increased. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integrity. The behavior of intracellular and extracellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the insect cell (Sf-9)/baculovirus expression system.

Carotenoid accumulation in the psychrotrophic bacterium Arthrobacter agilis in response to thermal and salt stress by N. Fong; M. Burgess; K. Barrow; D. Glenn (pp. 750-756).

A psychrotrophic strain of Arthrobacter agilis, isolated from Antarctic sea ice, grows from 5°C to 40°C and in culture media containing 0–10% (w/v) NaCl. Maximum growth rate occurred at 30–35°C with a drastic decline as the cultivation temperatures diverged. Adaptation to extremes of low temperature may be partially attributed to the production of the C-50 carotenoid bacterioruberin, and its glycosylated derivatives. Lowering of the cultivation temperature resulted in a concomitant increase in carotenoid production, which may contribute to membrane stabilisation at low temperature. Maximum biomass accumulation occurred at 5–30°C with a tenfold reduction at 40°C. Changes in growth rates were minimal in culture media containing 0–2% (w/v) NaCl at 10°C while a gradual decrease in growth rates occurred at higher salinity. Biomass accumulation at different salinity followed a trend similar to that observed with different cultivation temperatures. Maximum biomass accumulation was observed in culture media containing 0–5% (w/v) NaCl with a tenfold reduction at 10% (w/v) NaCl. Carotenoid production also decreased as salinity increased.

Bioconversion of nitriles by Candida guilliermondii CCT 7207 cells immobilized in barium alginate by J. Dias; R. Rezende; V. Linardi (pp. 757-761).

Nitrile degradation by Candida guilliermondii CCT 7207 using free and immobilized cell systems was compared. Different specific growth rates were observed for immobilized (µ _max=0.021 h^–1) and the free cells (µ _max=0.029 h^–1). The maximum specific rate of acetic acid formation was 0.387 h^–1 and 0.266 h^–1 for free and immobilized cells, respectively. Cell adhesion to the support materials was confirmed by scanning electron microscopy. When immobilized, the yeast was able to use high nitrile and amide concentrations (aliphatic and aromatic) as nitrogen sources. The results suggest that C. guilliermondii CCT 7207 presents a physiological pattern potentially useful for the bioremediation of polluted environments or for the bioproduction of amides and organic acid of high commercial value.

Xylanase from a newly isolated Fusarium verticillioides capable of utilizing corn fiber xylan by B. Saha (pp. 762-766).

A fungus, Fusarium verticillioides (NRRL 26518), was isolated by screening soil samples using corn fiber xylan as carbon source. The extracellular xylanase from this fungal strain was purified to apparent homogeneity from the culture supernatant by ultrafiltration using a 30,000 cut-off membrane, octyl-Sepharose chromatography and Bio gel A-0.5 m gel filtration. The purified xylanase (specific activity 492 U/mg protein; MW 24,000; pI 8.6) displayed an optimum temperature at 50 °C and optimum pH at 5.5, a pH stability range from 4.0 to 9.5 and thermal stability up to 50 °C. It hydrolyzed a variety of xylan substrates mainly to xylobiose and higher short-chain xylooligosaccharides. No xylose was formed. The enzyme did not require metal ions for activity and stability.

Redirection of pyruvate catabolism in Lactococcus lactis by selection of mutants with additional growth requirements by C. Henriksen; D. Nilsson (pp. 767-775).

Based on requirements for acetate or lipoic acid for aerobic (but not anaerobic) growth, Lactococcus lactis subsp. lactis mutants with impaired pyruvate catabolism were isolated following classical mutagenesis. Strains with defects in one or two of the enzymes, pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH) and the pyruvate dehydrogenase complex (PDHC) were obtained. Growth and product formation of these strains were characterized. A PFL-defective strain (requiring acetate for anaerobic growth) displayed a two-fold increase in specific lactate production compared with the corresponding wild-type strain when grown anaerobically. LDH defective strains directed 91–96% of the pyruvate towards α-acetolactate, acetoin and diacetyl production when grown aerobically in the presence of acetate and absence of lipoic acid (a similar characteristic was observed in an LDH and PDHC defective strain in the presence of both acetate and lipoic acid) and more than 65% towards formate, acetate and ethanol production under anaerobic conditions. Another strain with defective PFL and LDH was strictly aerobic. However, a variant with strongly enhanced diacetyl reductase activities (NADH/NAD⁺ dependent diacetyl reductase, acetoin reductase and butanediol dehydrogenase activities) was selected from this strain under anaerobic conditions by supplementing the medium with acetoin. This strain is strictly aerobic, unless supplied with acetoin.

Quantification of intracellular amino acids in batch cultures of Saccharomyces cerevisiae by M. Hans; E. Heinzle; C. Wittmann (pp. 776-779).

The dynamics of intracellular amino acid pools were determined in batch cultures of Saccharomyces cerevisiae. Immediate termination of metabolic activity was found to be necessary for accurate quantification of in vivo concentrations of intracellular amino acids, due to significant changes in most intracellular amino acid pools observed during extraction without an instantaneous stop of the metabolism. The method applied to batch-cultures of S. cerevisiae on glucose revealed complex dynamics in intracellular amino acid pools. The most drastic changes were observed during the diauxic shift and at the entry into the stationary phase. Even during phases of exponential growth on glucose and ethanol, cells showed significant variations in intracellular amino acid concentrations. The method presented can be used to investigate the physiology of yeast cultures, including industrially relevant batch and fed-batch processes.

Biomonitoring of continuous microbial community adaptation towards more efficient phenol-degradation in a fed-batch bioreactor by Benoît Guieysse; Per Wikström; Mats Forsman; Bo Mattiasson (pp. 780-787).

The anaerobic degradation of phenol was studied in a fed-batch culture. Nitrate was added as electron acceptor and phenol was provided three times, to a final concentration of 200 mg/l. Randomly amplified polymorphic DNA (RAPD) and terminal fraction fragment length polymorphism (T-RFLP) were used and compared in order to monitor the microbial succession in the reactor. Phenol degradation started after an initial lag phase of 14 days and was then completed within a few days. In addition, the duration of the lag phase was shortened and the degradation rate was increased after each phenol amendment. Nitrate reduction correlated with microbial growth and phenol depletion, confirming that the degradation was carried out anaerobically. Results from the DNA analysis showed that the structure of the microbial community changed after each phenol amendment. This study confirms the potential for anaerobic degradation of environmental pollutants and also confirms that microbial acclimation towards faster degradation rates occurred upon repeated substrate amendments. Furthermore, both of the DNA-based techniques described the phenol degradation-linked community shifts with similar general results. RAPD is a faster, simpler technique that gives a higher resolution and consequently reflects the shifts in the microbial community structure better, whereas T-RFLP is more suitable for phylogenetic studies.

Preliminary examinations for applying a carbazole-degrader, Pseudomonas sp. strain CA10, to dioxin-contaminated soil remediation by H. Habe; K. Ide; M. Yotsumoto; H. Tsuji; H. Hirano; J. Widada; T. Yoshida; H. Nojiri; T. Omori (pp. 788-795).

A method for bioremediation of chlorinated dibenzo-p-dioxins (CDDs) and dibenzofurans (CDFs) by a carbazole-utilizing bacterium, Pseudomonas sp. strain CA10, was developed. CA10 cells transferred to carbon- and nitrogen-free mineral medium supplemented with 1 mg carbazole (CAR)/ml grew rapidly during the first 2 days; and the cells at the end of this rapid growth period showed the highest 2,3-dichlorodibenzo-p-dioxin (2,3-Cl₂DD)-degrading activity. The CA10 cells pre-grown for 2 days efficiently degraded 2,3-Cl₂DD in aqueous solution at either 1 ppm or 10 ppm. The effect of inoculum density on the efficiency of 2,3-Cl₂DD degradation was investigated in a soil slurry microcosm [ratio of soil:water = 1:5 (w/v)]. The results showed that a single inoculation with CA10 cells at densities of 10⁷ CFU/g soil and 10⁹ CFU/g soil degraded 46% and 80% of 2,3-Cl₂DD, respectively, during the 7-day incubation. The rate of degradation of each CDD congener, 2-ClDD, 2,3-Cl₂DD, and 1,2,3-Cl₃DD (1 ppm each) by strain CA10 in the soil slurry system was not significantly influenced by the coexistence of the other congeners. Using this soil slurry system, we tried an experimental bioremediation of the actual dioxin-contaminated soil, which contained mainly tetra- to octochlorinated dioxins. Although the degradation rate of total CDD and CDF congeners by a single inoculation with CA10 cells was 8.3% after a 7-day incubation, it was shown that strain CA10 had a potential to degrade tetra- to hepta-chlorinated congeners including the most toxic compound, 2,3,7,8-tetrachlorinated dibenzo-p-dioxin.

Microbial degradation of phenanthrene and pyrene in a two-liquid phase-partitioning bioreactor by B. Guieysse; M. d. D.T.G. Cirne; B. Mattiasson (pp. 796-802).

A study was conducted to determine the potential of two-liquid phase-bioreactors for the treatment of (polycyclic aromatic hydrocarbons) PAHs. Phenanthrene and pyrene were supplied two times at a concentration of 100 mg/l of reactor broth, either as crystals or dissolved in silicone oil. Complete phenanthrene biodegradation was achieved within 3 days after each addition to the biphasic-inoculated reactor. Its concentration in the monophasic reactors dropped by 93% within 4 days, but remained incomplete for the duration of the experiment. Pyrene removal occurred to a limited extent only in the presence of phenanthrene. Significant pollutant losses were recorded in the monophasic reactors, most likely caused by volatilization. Pollutant degradation was improved upon repeated phenanthrene amendment to the biphasic system. Biphasic reactors allow the fast and complete degradation of PAHs and prevent their hazardous disappearance. The use of biphasic reactors for the degradation of poorly soluble pollutants should become more beneficial when the substrate-interface uptake mechanism is operating. Thus, biphasic reactors should be integrated into the microbial enrichment procedure.

Pyrene degradation by two fungi in a freshwater sediment and evaluation of fungal biomass by ergosterol content by C. Ravelet; C. Grosset; S. Krivobok; B. Montuelle; J. Alary (pp. 803-808).

Mucor racemosus var. sphaerosporus and Phialophora alba were investigated for their abilities to degrade pyrene in a freshwater sediment, with or without glucose supply as nutrient or carbon source, during 90 days. The ergosterol contents in sediment were quantified to estimate fungal biomass and to assess the correlation between fungal activity and biodegradation of pyrene. Results showed that, in an heterogeneous environment, these fungi presented different abilities to degrade pyrene. P. alba increased the degree of pyrene degradation by 9%, compared to the native micro-organisms, but a supply of glucose acted as an inhibitor to pyrene disappearance. M. racemosus var. sphaerosporus was not efficient at sediment bioremediation (with or without glucose added), because it reduced the rate of pyrene degradation by the native microflora. In any case, there was no increase of ergosterol in boxes during bioremediation experiments. In our experimental conditions, ergosterol content could not be correlated to pyrene degradation.

Development of a rapid pH-based biosensor to monitor and control the hygienic quality of reclaimed domestic wastewater by T. Dewettinck; K. Van Hege; W. Verstraete (pp. 809-815).

The re-use of treated domestic wastewater necessitates a rigorous control and rapid monitoring of the hygienic quality of the reclaimed water. For this purpose, a new pH-based biosensor was developed. The essence of the methodology of the sensor is the monitoring of the acidification due to bacterial metabolism of added glucose. To improve the sensitivity, the alkalinity of the water sample is reduced prior to monitoring the acidification. This is done by stripping CO₂ at a neutral or acidic pH value. The hygienic aspect of the sensor lies in the applied temperature (37 °C) and the use of N₂ as decarbonizing gas, thus creating conditions favorable for enteric bacteria. The developed sensor could be used on-site at an advanced stage of treatment, as an endpoint or intake quality control device. For both applications, a useful correlation was obtained between log total plate count and lag time or acidification rate, respectively. Absolute detection limits lay in the ranges of either 10³ colony-forming units (CFU)/ml in 6 h (endpoint quality control), or less than 10⁵ CFU/ml in 1 h (intake quality control).

A PCR test to identify Bacillus subtilis and closely related species and its application to the monitoring of wastewater biotreatment by P. Wattiau; M.-E. Renard; P. Ledent; V. Debois; G. Blackman; S. Agathos (pp. 816-819).

A PCR test based on the 16S rRNA gene was set up that could identify any of the five species of the 'Bacillus subtilis group' (B. subtilis, B. pumilus, B. atrophaeus, B. licheniformis and B. amyloliquefaciens). The test was directly applicable to single colonies and showed excellent specificity. In the mixed population context of wastewater analysis, direct detection of the target Bacillus species by PCR on either crude or purified DNA extracts had poor sensitivity. When assayed on cell suspensions derived from enriched wastewater samples, sensitivity was increased. Using a simple calibration method, it was possible to estimate the proportion of the target organisms. This method was found suitable for easy monitoring of a wastewater bioaugmentation experiment carried out with a mixture of sporulated Bacillus strains.

Aerobic treatment of a concentrated urea wastewater with simultaneous stripping of ammonia by K. Rittstieg; K.-H. Robra; W. Somitsch (pp. 820-825).

An industrial wastewater containing a total Kjeldahl nitrogen (TKN) of 12.80 g l^–1 was treated in a continuously fed activated sludge reactor. The main contaminant was urea (21.52 g l^–1), together with minor amounts of the nitrification inhibitor dicyandiamide (0.46 g l^–1) and free ammonia (0.56 g l^–1). The wastewater was diluted 1:1 with water and treated under alkaline conditions (pH 9.4), enabling the simultaneous hydrolysis of urea and stripping of free ammonia in one aerobic reactor. Experiments were conducted to eliminate the remaining ammonia in a separate treatment unit by nitrification/denitrification. An adapted nitrifying bacterial population was isolated which was able to nitrify at a rate of 0.1 g nitrogen l^–1 day^–1 at a dicyandiamide concentration of 0.22 g l^–1. However, this was found to be too slow for an industrial-scale operation. Therefore, separate stripping with air or steam after pH adjustment to ≥10.5 is proposed. The diluted wastewater was treated with a hydraulic retention time of 6 days, corresponding to a volumetric nitrogen loading rate of 1.1 g nitrogen l^–1 day^–1 with an overall TKN reduction of 78.0%.

Investigations into the application of a process for the determination of microbial activity in biofilms by D. Holtmann; D. Sell (pp. 826-830).

The formation of biofilms in a waste paper medium was studied in a pilot plant by analysing the redox potential in the biofilm. Miniaturised redox electrodes were applied at the reactor wall/biofilm phase boundary. With this measurement set-up, it was possible to demonstrate the effectiveness of biocides and thus to avoid under- and over-doses with these agents. The redox signals measured were correlated with reference methods, such as colony-forming units and dehydrogenase activity.

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Applied Microbiology and Biotechnology (v.56, #5-6)