Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.59, #2-3)

Drugs from the seas – current status and microbiological implications by P. Proksch; R. Edrada; R. Ebel (pp. 125-134).

The oceans are the source of a large group of structurally unique natural products that are mainly accumulated in invertebrates such as sponges, tunicates, bryozoans, and molluscs. Several of these compounds (especially the tunicate metabolite ET-743) show pronounced pharmacological activities and are interesting candidates for new drugs primarily in the area of cancer treatment. Other compounds are currently being developed as an analgesic (ziconotide from the mollusc Conus magus) or to treat inflammation. Numerous natural products from marine invertebrates show striking structural similarities to known metabolites of microbial origin, suggesting that microorganisms (bacteria, microalgae) are at least involved in their biosynthesis or are in fact the true sources of these respective metabolites. This assumption is corroborated by several studies on natural products from sponges that proved these compounds to be localized in symbiotic bacteria or cyanobacteria. Recently, molecular methods have successfully been applied to study the microbial diversity in marine sponges and to gain evidence for an involvement of bacteria in the biosynthesis of the bryostatins in the bryozoan Bugula neritina.

Industrial processes with animal cells by G. Kretzmer (pp. 135-142).

Industrial processes involving animal cells for the production of useful products still seem to be rather uncommon. Nevertheless, during the last four decades of the last century the number of relevant processes has increased from production of virus vaccines to monoclonal antibodies and finally complex structured glycoproteins. As soon as cell lines became permanent and culture medium changed from purely biological fluids to more or less defined chemical media, large-scale cultivation could begin. The developments of the 1970s – fusion of cells to form hybridomas, and genetic engineering – triggered a second wave of products. Monoclonal antibodies and recombinant proteins for diagnosis and therapy set new challenges for the inventors. Historically, there has been no straightforward process development since the product dictates the process operation. Therefore, the scale of production covers the whole range from small multiple-unit reactors (flasks or roller bottles) up to 10,000-l single-unit batch reactors. Products with high value and small demand can be produced in multiple-unit systems whereas "bulk" products for vaccination and therapy may need large-scale bioreactors to be cost effective. All the different systems have their advantages and disadvantages and significant challenges that curb the development of effective perfusion cultures still remain.

Biotechnology and bioremediation: successes and limitations by M. Dua; A. Singh; N. Sethunathan; A. Johri (pp. 143-152).

With advances in biotechnology, bioremediation has become one of the most rapidly developing fields of environmental restoration, utilizing microorganisms to reduce the concentration and toxicity of various chemical pollutants, such as petroleum hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, phthalate esters, nitroaromatic compounds, industrial solvents, pesticides and metals. A number of bioremediation strategies have been developed to treat contaminated wastes and sites. Selecting the most appropriate strategy to treat a specific site can be guided by considering three basic principles: the amenability of the pollutant to biological transformation to less toxic products (biochemistry), the accessibility of the contaminant to microorganisms (bioavailability) and the opportunity for optimization of biological activity (bioactivity). Recent advances in the molecular genetics of biodegradation and studies on enzyme-tailoring and DNA-shuffling are discussed in this paper.

A new photobioreactor for continuous marennin production with a marine diatom: influence of the light intensity and the immobilised-cell matrix (alginate beads or agar layer) by T. Lebeau; P. Gaudin; R. Moan; J.-M. Robert (pp. 153-159).

In oyster ponds, the marine diatom Haslea ostrearia synthesises and excretes a hydrosoluble pigment of commercial interest called marennin. During the benthic stage, when algal cells are naturally immobilised in their own polysaccharides, marennin production is higher. To optimise this production, axenic cultures of H. ostrearia were immobilised in a polysaccharidic matrix (alginate or agar) and introduced into a new photobioreactor device for continuous marennin production. Solute diffusion was improved using an alginate beads monolayer, leading to higher levels of cell growth (a 2-fold higher cell concentration) and marennin productivity (7.57–8.80 mg day^–1 l^–1). An increase in the light intensity (from 3.0 to 8.5×10¹⁶ quanta cm^–2 s^–1) led to an earlier and 1.3-fold higher production of marennin. However, the higher light intensity led to a higher rate of cell death [0.29 instead of 0.40 ng chlorophyll a (10⁶ cells)^–1]. Due to the secondary nature of marennin metabolism, it would be necessary to alternate between culture conditions favouring cell growth (moderate light intensity and no limiting nitrate supply) and those promoting marennin production (high light intensity and limiting nitrate supply).

Thermophilic production of lactic acid using integrated membrane bioreactor systems coupled with monopolar electrodialysis by H. Danner; L. Madzingaidzo; C. Thomasser; M. Neureiter; R. Braun (pp. 160-169).

The thermophilic Bacillus strain BS119 was selected for this study to demonstrate the long term performance of lactic acid production and simultaneous pre-purification. Integrated continuous cell recycle cultivation using ultra-filtration membrane bioreactor (MBR) systems was investigated. The permeate from the MBR was routed to an on-line electrodialysis (ED) to recover, pre-purify and concentrate lactate. The cultivation and ED was operated at 60°C for more than 1,000 h at a pH of 6.5. At lower dilution rate (0.02 h^–1), lactate concentration reached a maximum of 55 g l^–1 with clearly lower residual glucose levels. At 0.04 h^–1, lactate concentration was significantly lower at 35 g l^–1. Maximal volumetric productivities of 1.38 g l^–1 h^–1 were achieved. Under stable conditions, lactic acid yield on consumed glucose appeared stable at around 80%. It could be demonstrated that the addition of supplements like yeast extract and peptone severely influences product formation. Integration of mono-polar ED with the MBR systems yields lactate solutions with concentrations of up to 115 g l^–1. Because of the low substrate feed concentrations (less than 50 g l^–1), lactate flux was rather poor, reaching a low maximum of 140 g m^–2 h^–1; nevertheless, stack energy consumption was positive with an average of 0.49 kWh kg^–1 lactate.

Coupled lactic acid fermentation and adsorption by C. Chen; L.-K. Ju (pp. 170-174).

Polyvinylpyridine (PVP) and activated carbon were evaluated for coupled lactic acid fermentation and adsorption, to prevent the product concentration from reaching inhibitory levels. The lactic acid production doubled as a result of periodical circulation of the fermentation broth through a PVP adsorption column. The adsorbent was then regenerated and the adsorbed lactate harvested, by passing 0.1 N NaOH through the column. However, each adsorption–regeneration cycle caused about 14% loss of the adsorption capacity, thus limiting the practical use of this rather expensive adsorbent. Activated carbon was found much more effective than PVP in lactic acid and lactate adsorption. The cells of Lactobacillus delbrueckii subsp. delbrueckii (LDD) also had strong tendency to adsorb on the carbon. A study was therefore conducted using an activated carbon column for simultaneous cell immobilization and lactate adsorption, in a semi-batch process with periodical medium replacement. The process produced lactate steadily at about 1.3 g l^–1h^–1 when the replacement medium contained at least 2 g l^–1 of yeast extract. The production, however, stopped after switching to a medium without yeast extract. Active lactic acid production by LDD appeared to require yeast extract above a certain critical level (<2 g l^–1).

Inoculum production of the ectomycorrhizal fungus Pisolithus microcarpus in an airlift bioreactor by M. Rossi; J. Souza; V. Oliveira (pp. 175-181).

Many important tree species in reforestation programs are dependent on ectomycorrhizal symbiosis in order to survive and grow, mainly in poor soils. The exploitation of this symbiosis to increase plant productivity demands the establishment of inoculum production methods. This study aims to propose an inoculum production method of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) using liquid fermentation in an airlift bioreactor with external circulation. The fungus grew as dark dense pellets during a batch fermentation at 25.5°C and air inlet of 0.26–0.43 vvm. The maximum biomass (dry weight) achieved in the airlift bioreactor was approximately 5 g.l^–1 after 10–11 days. The specific growth rate (µ_x) in the exponential phase was 0.576 day^–1, the yield factor (Y_X/S) 0.418, and the productivity (P_X) 0.480 g.l^–1.day^–1. This specific growth rate was higher than that observed by other authors during fermentation processes with other Pisolithus isolates. The method seems to be very suitable for biomass production of this fungus. However, new studies on the fungus growth morphology in this system, as well as on the efficiency of the process for the cultivation of other ectomycorrhizal fungi, are necessary. It is also necessary to test the infectivity and efficiency of the inoculum towards the hosts.

Dietary-fiber-degrading enzymes from a human intestinal Clostridium and their application to oligosaccharide production from nonstarchy polysaccharides using immobilized cells by N. Nakajima; K. Ishihara; Y. Matsuura (pp. 182-189).

The secretion of nonstarchy polysaccharide-degrading enzymes from an anaerobic human intestinal bacterium, Clostridium butyricum-beijerinckii (isolated from human feces), was investigated. Growth of the bacterium was found when laminarin, konjac glucomannan, and pectic acid were added separately to the culture media as sole carbon source. The corresponding degrading enzymes for these dietary fibers, laminarinase (endo-1,3-β-glucanase), endo-1,4-β-mannanase, endo- and exo-pectate lyases, and pectin methylesterase, were then purified and characterized. These extracelluar enzymes, which were secreted by the bacterium in the human large intestine, were considered to contribute to digestion of the ingested dietary fibers to their oligosaccharides, following by short-chain fatty acid fermentation by the bacterium. We have developed cell immobilization techniques of the bacterium on cellulose-foam carriers that are effective for continuous production of the oligosaccharides from the dietary fibers in a fed-batch reactor system. From 9 g of pectic acid, a total of 3.96 g of 4,5-unsaturated digalacturonic acid was produced over 40 h in four 500-ml batchcultures. In the same manner, the corresponding oligosaccharides were obtained from konjac glucomannan and laminarin with average conversion rates of around 30–40%.

Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis by A. Tye; F. Siu; T. Leung; B. Lim (pp. 190-197).

A novel phytase gene (phyL) was cloned from Bacillus licheniformis by multiple steps of degenerate and inverse PCR. The coding region of the phyL gene was 1,146 bp in size and a promoter region of approximately 300 bp was identified at the upstream sequence. This gene, together with a phytase gene (168phyA) identified in the B. subtilis strain 168 genome by a homology search, was cloned and over-expressed in B. subtilis using a φ105MU331 prophage vector system. Up to 35 units of phytase/ml were secreted into the culture media; and mature enzymes of around 44–47 kDa were purified for characterization. Both phytases exhibited broad temperature and pH optima and showed high thermostability. Of the two, the phytase encoded by phyL exhibited higher thermostability, even at a lower calcium concentration, as it was able to recover 80% of its original activity after denaturation at 95 °C for 10 min. With their neutral pH optima and good temperature stabilities, these Bacillus phytases are good candidates for animal feed applications and transgenic studies.

Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces by L.-L. Kiiskinen; L. Viikari; K. Kruus (pp. 198-204).

A novel laccase from the ascomycete Melanocarpus albomyces was purified and characterised. The enzyme was purified using anion exchange chromatography, hydrophobic interaction chromatography and gel filtration, and the purified laccase was biochemically characterised. It had activity towards typical substrates of laccases including 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate), dimethoxyphenol, guaiacol, and syringaldazine. The laccase showed good thermostability and it had a pH optimum at neutral pH, both unusual properties for most known fungal laccases. The activity of the laccase from M. albomyces was highest at 60–70°C. With guaiacol and syringaldazine the pH optima were rather broad: 5–7.5 and 6–7, respectively. It retained 50% of its activity after 5 h incubation at 60°C. The molecular weight of the laccase was about 80 kDa and the isoelectric point 4.0. The ultraviolet-visible absorption and electron paramagnetic resonance spectra of the purified laccase indicated that the typical three types of copper were present.

Influence of threonine exporters on threonine production in Escherichia coli by D. Kruse; R. Krämer; L. Eggeling; M. Rieping; W. Pfefferle; J. Tchieu; Y. Chung; M. Saier; A. Burkovski (pp. 205-210).

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [¹⁴C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.

Construction of self-disruptive Bacillus megaterium in response to substrate exhaustion for polyhydroxybutyrate production by K. Hori; M. Kaneko; Y. Tanji; X.-H. Xing; H. Unno (pp. 211-216).

In order to establish a novel recovery system for polyhydroxyalkanoates, a self-disruptive strain of Bacillus megaterium that responds to substrate exhaustion was constructed. A gene cassette carrying the lysis system of Bacillus amyloliquefaciens phage – holin and endolysin – was inserted into the Escherichia coli-Bacillus subtilis shuttle vector pX under the control of a xylose-inducible expression system, xylR-xylA ′. In this system, the expression of a target gene is induced by xylose but inhibited by glucose, which acts as an anti-inducer. B. megaterium was transformed with pX conveying the phage lysis system, which was integrated into the amyE locus of chromosomal DNA of B. megaterium by homologous recombination. The lysis system caused self-disruption of the transformant cells effectively even when expression of the lysis genes was induced during stationary phase. For the production of polyhydroxybutyrate (PHB), the transformant was grown in a medium containing glucose as a substrate in the presence of xylose. When the glucose concentration approached zero, self-disruption was spontaneously induced, releasing intracellularly accumulated PHB into the culture broth. This system realizes timely cell disruption immediately after the PHB content in the cell reaches a maximum level.

Bioengineered emulsans from Acinetobacter calcoaceticus RAG-1 transposon mutants by A. Johri; W. Blank; D. Kaplan (pp. 217-223).

Transposon mutants of Acinetobacter calcoaceticus strain RAG-1 were studied in an effort to control fatty acid (FA) substitution patterns of emulsan, a bioemulsifier secreted by the organism. The disrupted genes, involved in the biosynthetic pathways of biotin, histidine, cysteine or purines, influenced the level and types of FAs incorporated into emulsan. The structural variants of emulsan generated by the transposon mutants were characterized for yield, FA content, molecular weight, and emulsification behavior when grown on a series of FAs of different chain lengths from C11 to C18. Yields of emulsan from the transposon mutants were found to be lower than the parent strain and depended on the type of FA used to supplement the growth medium. Mutants 13D (His–) and 52D (Cys–) grown on LB plus C16 or C14, respectively, exhibited enhanced emulsifying activity compared to A. calcoaceticus RAG-1. The presence and composition of long chain FAs on the polysaccharide backbone influenced emulsification behavior: particularly a high mole percentage of C16 (48%) and C18 (42%). The results provide important insight into the bioengineering of bioemulsifier-producing microorganisms and provide a path towards highly tailored novel amphipathic structures to utilize as biodegradable in environmental, biomedical, and personal care applications.

Metabolic engineering of Saccharomyces cerevisiae for production of novel lipid compounds by J. Dyer; D. Chapital; J. Kuan; R. Mullen; A. Pepperman (pp. 224-230).

The yeast Saccharomyces cerevisiae has been modified successfully for production of numerous metabolites and therapeutic proteins through metabolic engineering, but has not been utilized to date for the production of lipid-derived compounds. We developed a lipid metabolic engineering strategy in S. cerevisiae based upon culturing techniques that are typically employed for studies of peroxisomal biogenesis; cells were grown in media containing fatty acids as a sole carbon source, which promotes peroxisomal proliferation and induction of enzymes associated with fatty acid β-oxidation. Our results indicate that growth of yeast on fatty acids such as oleate results in extensive uptake of these fatty acids from the media and a subsequent increase in total cellular lipid content from 2% to 15% dry cell weight. We also show that co-expression of plant fatty acid desaturases 2 and 3 (FAD2 and FAD3), using a fatty acid-inducible peroxisomal gene promoter, coupled the processes of fatty acid uptake with the induction of a new metabolic pathway leading from oleic acid (18:1) to linolenic acid (18:3). Finally, we show that cultivation of yeast cells in the presence of triacylglycerols and exogenously supplied lipase promotes extensive incorporation of triglyceride fatty acids into yeast cells. Collectively, these results provide a framework for bioconversion of low-cost oils into value-added lipid products.

Introduction of the carbohydrate-activated promoter P_malK for recombinant protein production by M. Boström; G. Larsson (pp. 231-238).

A production protocol for the use of the malK promoter was established. The protocol includes two phases: an initial fed-batch phase on glucose to reach a high cell density and a fed-batch phase on maltose for production of the desired recombinant protein. It is suggested that this cultivation scheme could be used for all promoters that are catabolite repressed by glucose and where growth and production need to be separated. The specific feature of this system is shown by its ability to control the rate of synthesis of the product protein, ß-galactosidase. In the production phase with a constant feed or an exponential feeding of 0.1 h^–1 it took 4 h longer to reach the maximum specific production rate than with the higher dilution rates of 0.25 h^–1 and 0.4 h^–1, respectively. In the above experiments a dilution rate of 0.3 h^–1 in the growth phase was used. The volumetric production of this system could furthermore be extended to 40 h. All protocol procedures so far tested resulted in the same maximum production rate, but reached in different lengths of time. It is argued that this system is particularly well suited for the production of proteins that have a complex structure and/or need to be produced in a soluble form or to be exported to the periplasm.

High-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter by E. Toksoy; Z. Önsan; B. Kirdar (pp. 239-245).

Three different expression systems were constructed for the high-level production of TaqI restriction endonuclease in recombinant Escherichia coli cells. In system [R], the TaqI endonuclease gene was cloned and expressed under the control of the strong T7 RNA polymerase promoter. To protect cellular DNA, methylase protection was provided by constitutive co-expression of TaqI methylase activity either by cloning the TaqI methylase gene on a second plasmid (system [R,M]) or by constructing a recombinant plasmid harboring both the endonuclease and methylase genes (system [R+M]). In batch shake flasks containing complex media, co-expression of the methylase gene in systems [R,M] and [R+M] resulted in a 2- and 3-fold increase in volumetric productivity over system [R], yielding activities of 250×10⁶ U l^–1 and 350×10⁶ U l^–1, which were 28 and 39 times higher than the data in the literature, respectively. Under controlled bioreactor conditions in chemically defined medium, co-expression of methylase activity greatly improved the yield and specific TaqI endonuclease productivity of the recombinant cells, and reduced acetic acid excretion levels. System [R,M] is preferable for high expression levels at longer operation periods, while system [R+M] is well-suited for high expression levels in short-term bioreactor operation.

Expression of a gene for Mn-peroxidase from Coriolus versicolor in transgenic tobacco generates potential tools for phytoremediation by Y. Iimura; S. Ikeda; T. Sonoki; T. Hayakawa; S. Kajita; K. Kimbara; K. Tatsumi; Y. Katayama (pp. 246-251).

In efforts aimed at the detoxification of contaminated areas, plants have many advantages over bacteria and fungi. We are attempting to enhance the environmental decontamination functions of plants by transferring relevant genes from microorganisms. When the gene for Mn-peroxidase (MnP) from Coriolus versicolor was expressed in transgenic tobacco plants, one line (designated fMnP21) expressed MnP activity at levels 54-fold higher than in control lines. When undamaged roots of transgenic plants were applied to liquid medium supplemented with 250 µM pentachlorophenol (PCP), the decrease in the level of PCP in fMnP21 (86% reduction) was about 2-fold higher than that in control lines (38% reduction). Expression of the gene for MnP in the transgenic plants had no obvious negative effects on their vegetative and sexual growth. Our system should contribute to the development of novel methods for the removal of hazardous chemicals from contaminated environments using transgenic plants.

Production of active bovine cathepsin C (dipeptidyl aminopeptidase I) in the methylotrophic yeast Candida boidinii by T. Komeda; K. Tazumi; H. Shimada; K. Kano; T. Hayashi; H. Saito; H. Tsumura; N. Kato; Y. Sakai; K. Kondo (pp. 252-258).

The heterologous production of active bovine cathepsin C (CTC; dipeptidyl aminopeptidase I) was investigated. Attempts to express CTC in Escherichia coli were hampered by formation of inclusion bodies that were partially degraded. To overcome this impediment, secretion of recombinant CTC was attempted in the methylotrophic yeast Candida boidinii. A DNA fragment encoding bovine procathepsin C was synthesized based on preferred codon usage in C. boidinii and placed downstream of the C. boidinii proteinase A signal sequence resulting in secretion of active CTC into the culture medium. The gene was expressed under the control of the methanol-inducible formate dehydrogenase gene promoter. Production levels were significantly improved by using a protease-deficient strain, changing medium composition, and by lowering the temperature of induction. When the recombinant C. boidinii was grown for 90 h in a jar-fermenter, active CTC was secreted with a yield of up to approximately 12 mg/l.

Cell surface-engineered yeast with ability to bind, and self-aggregate in response to, copper ion by K. Kuroda; M. Ueda; S. Shibasaki; A. Tanaka (pp. 259-264).

In order to construct a cell surface-engineered yeast Saccharomyces cerevisiae that facilitates adsorption and recovery of heavy metal ions, we endowed it with the ability to self-aggregate in response to binding and accumulation of copper ion. A fusion gene for the expression of GTS1, which encodes a putative zinc-finger transcription factor related to occurrence of cell-aggregation, was constructed under the control of the copper ion-inducible CUP1 promoter from the yeast metallothionein gene. The multicopy plasmid carrying the fusion gene was introduced into a cell surface-engineered yeast displaying histidine hexa-peptide, which can chelate copper ion. This transformant strain aggregated in medium only in the presence of copper ion, with aggregation induced by as little as 1 mM copper ion. The copper ion-induced aggregation did not interfere with the copper ion-adsorbing function of the cell surface-engineered yeast, indicating that this transformant strain has the twin features of enhanced cell surface adsorption of copper ion and self-aggregation in response to environmental copper ion.

Sinorhizobium fredii isolates can be specifically identified by a 260 bp fragment from the nolXWBTUV locus by L. Videira; G. Pastorino; V. Martinez Alcántara; P. Balatti (pp. 265-269).

A pair of primers homologous to the nolXWBTUV locus generated a 260 bp fragment by PCR only in the presence of Sinorhizobium fredii template DNA of different quality. This resulted in a fast and accurate method for the identification of S. fredii either from pure DNA, whole bacterial cells or nodule extracts. By means of two PCR fragments, one specific for S. fredii (260-bp) and the other specific for Bradyrhizobium japonicum (RSα), we found that S. fredii strain SMH12 and B. japonicum E109 were equally efficient at developing nodules on soybean plants grown under controlled environmental conditions.

Nitrate regulation of α-aminoadipate reductase formation and lysine inhibition of its activity in Penicillium chrysogenum and Acremonium chrysogenum by M. Hijarrubia; J. Aparicio; J. Martín (pp. 270-277).

α-Aminoadipate reductase (α-AAR) is a key enzyme in the branched pathway for lysine and β-lactam biosynthesis of filamentous fungi since it competes with α-aminoadipyl-cysteinyl-valine synthetase for their common substrate L-α-aminoadipic acid. The α-AAR activity in two penicillin-producing Penicillium chrysogenum strains and two cephalosporin-producing Acremonium chrysogenum strains has been studied. The α-AAR activity peaked during the growth-phase preceding the onset of antibiotic production, which coincides with a decrease in α-AAR activity, and was lower in high penicillin- or cephalosporin-producing strains. The α-AAR required NADPH for enzyme activity and could not use NADH as electron donor for reduction of the α-aminoadipate substrate. The α-AAR protein of P. chrysogenum was detected by Western blotting using anti-α-AAR antibodies. The mechanism of lysine feedback regulation in these two filamentous fungi involves inhibition of the α-AAR activity but not repression of its synthesis by lysine. This is different from the situation in yeasts where lysine feedback inhibits and represses α-AAR. Nitrate has a strong negative effect on α-AAR formation as shown by immunoblotting studies of α-AAR. The nitrate effect was reversed by lysine.

Effect of medium composition, flow rate, and signaling compounds on the formation of soluble extracellular materials by biofilms of Chromobacterium violaceum by D. Martinelli; R. Bachofen; H. Brandl (pp. 278-283).

Biofilms of the homoserine-lactone-defective strain Chromobacterium violaceum CV026 were grown on silicone surfaces in a flow chamber. The effect of medium flow rate, different levels of N-butanoyl-homoserine lactone, and nutrients on biofilm activity was studied by quantifying the proteins and exopolysaccharides attached to the cells. To compare the effect of each of the three variables within a wide range, the experiments were designed as a full factorial search with three levels for each variable. Calculated contour plots demonstrated that N-acyl homoserine lactone is an important determinant of both the amount and the composition of the compounds excreted into the medium.

Degradation of chlorophenols by Phanerochaete chrysosporium: effect of 3,4-dichlorophenol on extracellular peroxidase activities by R. Duran; C. Deschler; S. Precigou; P. Goulas (pp. 284-288).

Extracellular peroxidases play an important role in the degradation of chlorophenols by Phanerochaete chrysosporium. Depending on the moment of 3,4-dichlorophenol addition, the production of lignin peroxidase and manganese peroxidase in C-limited agitated cultures was affected in opposite ways. In cultures that received 3,4-dichlorophenol at the time of inoculation, fungal growth was reduced and peroxidases were not produced, whereas peroxidase activities were stabilized after 3,4-dichlorophenol addition to pregrown cultures. Further investigation revealed that mRNA encoding lignin peroxidase was not produced in cultures started with 3,4-dichlorophenol, suggesting that the onset of secondary metabolism was affected. In addition, the stabilization of lignin peroxidase activity was not the result of an activation of lignin peroxidase gene transcription, as shown by Northern blot experiments, but likely due to the inhibition of peroxidase degradation by extracellular proteases.

Effect of glucose on glycerol bioconversion by Lactobacillus reuteri by Q. Lüthi-Peng; F. Dileme; Z. Puhan (pp. 289-296).

The impact of glucose on glycerol metabolism, especially on 3-hydroxypropionaldehyde (3-HPA) accumulation by resting cells of Lactobacillus reuteri has been investigated. Two systems were used in the study: MRS^– (modified MRS – omitting glucose, acetate and Tween 80) and distilled water (H₂O). In MRS^–, addition of glucose enhanced glycerol metabolism in resting cells of L. reuteri, consequently increasing the accumulation of 3-HPA by regulating the NAD/NADH ratio. Enhanced glycerol metabolism correlated positively with the concentration of glucose. NADH produced during glucose metabolism was preferentially reoxidized to NAD by the reduction of 3-HPA to 1,3-propanediol; an adequate supply of glycerol therefore outweighed the repression of glucose on the accumulation of 3-HPA. At a molar ratio of glucose to glycerol no greater than 0.33, accumulation of 3-HPA was favored. In non-growing medium (H₂O), addition of glucose seemed to be counter-productive with respect to 3-HPA accumulation. Lactate had a positive impact on glycerol metabolism, presumably by altering the redox flux, resulting in enhanced 3-HPA accumulation in both MRS^– and H₂O systems.

Quantitative determination of the spatial distribution of pure- and mixed-strain immobilized cells in gel beads by immunofluorescence by Y. Doleyres; I. Fliss; C. Lacroix (pp. 297-302).

A new method was developed to detect and quantify two strains, Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707, immobilized separately and co-immobilized in gel beads, using specific polyclonal antibodies and confocal laser-scanning microscopy. The establishment of biomass concentration profiles for each strain was measured during colonization of beads using successive pH-controlled batch fermentations. Growth occurred preferentially in 200- and 300-µm peripheral layers of the beads for L. diacetylactis and B. longum, respectively. Repeated-batch cultures with immobilized cells permitted the production of a mixed culture containing a non-competitive strain of bifidobacteria, as a result of immobilized-cell growth and high cell-release activity from the beads. During co-immobilized fermentations, there were no apparent interactions between the strains.

Use of carbon and energy balances in the study of the anaerobic metabolism of Enterobacter aerogenes at variable starting glucose concentrations by A. Converti; P. Perego (pp. 303-309).

The anaerobic metabolism of Enterobacter aerogenes was studied in batch culture at increasing initial glucose levels (9.0<S _o <72 g l^–1). The ultimate concentrations of fermentation products were utilized to check a metabolic flux analysis based on simple carbon mass and energy balances that promise to be suitable for the study of different fermentation processes, either under aerobic or anaerobic conditions. The stoichiometric coefficients of products collected at increasing starting glucose concentrations under anaerobic conditions suggest: (a) little influence of starting glucose level on the formation of the main fermentation products (2,3-butanediol and ethanol); (b) possible inhibition of 2,3-butanediol and lactate formations by increased ethanol concentration; (c) consequent increase in carbon flux through the remaining metabolic pathways with increased molar productions of succinate, acetate and hydrogen; (d) relative constancy of the molar production of ATP and CO₂.

Fed-batch cultivation of baker's yeast followed by nitrogen or carbon starvation: effects on fermentative capacity and content of trehalose and glycogen by H. Jørgensen; L. Olsson; B. Rønnow; E. Palmqvist (pp. 310-317).

An industrial strain of Saccharomyces cerevisiae (DGI 342) was cultivated in fed-batch cultivations at a specific growth rate of 0.2 h^–1. The yeast was then exposed to carbon or nitrogen starvation for up to 8 h, to study the effect of starvation on fermentative capacity and content of protein, trehalose and glycogen. Nitrogen starvation triggered the accumulation of trehalose and glycogen. After 8 h of starvation, the content of trehalose and glycogen was increased 4-fold and 2-fold, respectively. Carbon starvation resulted in a partial conversion of glycogen into trehalose. The trehalose content increased from 45 to 64 mg (g dry-weight)^–1, whereas the glycogen content in the same period was reduced from 55 to 5 mg (g dry-weight)^–1. Glycogen was consumed faster than trehalose during storage of the starved yeast for 1 month. Nitrogen starvation resulted in a decrease in the protein content of the yeast cells, and the fermentative capacity per gram dry-weight decreased by 40%. The protein content in the carbon-starved yeast increased as a result of starvation due to the fact that the content of glycogen was reduced. The fermentative capacity per gram dry-weight was, however, unaltered.

Alkanotrophic Rhodococcus ruber as a biosurfactant producer by J. Philp; M. Kuyukina; I. Ivshina; S. Dunbar; N. Christofi; S. Lang; V. Wray (pp. 318-324).

In this report we examined the structure and properties of surface-active lipids of Rhodococcus ruber. Most historical interest has been in the glycolipids of Rhodococcus erythropolis, which have been extensively characterised. R. erythropolis has been of interest due to its great metabolic diversity. Only recently has the metabolic potential of R. ruber begun to be explored. One major difference in the two species is that most R. ruber strains are able to oxidise the gaseous alkanes propane and butane. In preparation for investigation of the effects of gas metabolism on biosurfactant production, we set out to characterise the biosurfactants produced during growth on liquid n-alkanes and to compare these with R. erythropolis glycolipids.

Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain by Y. Tanaka; T. Matsui; J. Konishi; K. Maruhashi; R. Kurane (pp. 325-328).

Rhodococcus sp. KT462, which can grow on either benzothiophene (BT) or dibenzothiophene (DBT) as the sole source of sulfur, was newly isolated and characterized. GC and GC-MS analyses revealed that strain KT462 has the same BT desulfurization pathway as that reported for Paenibacillus sp. A11–2 and Sinorhizobium sp. KT55. The desulfurized product of DBT produced by this strain, as well as other DBT-desulfurizing bacteria such as R. erythropolis KA2-5-1 and R. erythropolis IGTS8, was 2-hydroxybiphenyl. A resting cells study indicated that this strain was also able to degrade various alkyl derivatives of BT and DBT.

An arming yeast with the ability to entrap fluorescent 17β-estradiol on the cell surface by M. Yasui; S. Shibasaki; K. Kuroda; M. Ueda; N. Kawada; J. Nishikawa; T. Nishihara; A. Tanaka (pp. 329-331).

We constructed a novel surface-engineered yeast displaying the ligand-binding domain of the rat estrogen receptor (ERLBD). ERLBD, display of which on the yeast cell surface was confirmed by immunofluorescence, possessed strong binding activity to fluorescent 17β-estradiol – an analogue of the natural ligand of the estrogen receptor – that was comparable to the activity of the native receptor. Environmental homeostasis has recently been disturbed by endocrine disruptors, which cause confusion in the hormone secretion system. It is therefore very important to identify chemical compounds with hormone-like activity and remove them from the environment. The present results demonstrate that the new arming yeast displaying ERLBD on its cell surface will be capable of screening, entrapping, and removing estradiol-like compounds from the environment.

Hydraulic selection pressure-induced nitrifying granulation in sequencing batch reactors by J.-H. Tay; S.-F. Yang; Y. Liu (pp. 332-337).

The effect of hydraulic selection pressure on the development of nitrifying granules was investigated in four column-type sequencing batch reactors (SBR). The nature of SBR is cycle operation, thus SBR cycle time can serve as a main hydraulic selection pressure imposed on the microbial community in the system. No nitrifying granulation was observed in the SBR operated at the longest cycle time of 24 h, due to a very weak hydraulic selection pressure, while the washout of nitrifying sludge was found in the SBR run at the shortest cycle time of 3 h, and led to a failure of nitrifying granulation. Excellent nitrifying granules with a mean diameter of 0.25 mm and specific gravity of 1.014 were developed in a SBR operated at cycle times of 6 h and 12 h, respectively. The results further showed that a short cycle time would stimulate microbial activity, production of cell polysaccharides and also improve the cell hydrophobicity. These hydraulic selection pressure-induced microbial changes favour the formation of nitrifying granules. This work, probably for the first time, shows that nitrifying granules can be developed at a proper hydraulic selection pressure in terms of SBR cycle time. Nitrifying granulation is a novel biotechnology which has a great potential for wastewater nitrification.

Effects of nitrite and ammonium on methane-dependent denitrification by M. Waki; Y. Tanaka; T. Osada; K. Suzuki (pp. 338-343).

For effective application of methane-dependent denitrification (MDD) in the treatment of wastewater containing NO₂ ^– or NH₄ ⁺, the effect of these inorganic nitrogen compounds on MDD activity needs to be clarified. The MDD activity of sludge acclimatized with CH₄ and O₂ was determined with mineral media of different nitrogen-compound compositions in the presence of 0.21 atm CH₄ and 0.20 atm O₂. Incubations with media containing only NO₂ ^– or two of the three inorganic nitrogen compounds (NO₃ ^–+NO₂ ^–, NO₂ ^–+NH₄ ⁺ or NH₄ ⁺+NO₃ ^–) resulted in MDD activity equal to or higher than that with media containing only NO₃ ^–. However, there was no MDD activity in media containing NO₂ ^– at 10 °C, probably because of serious inhibition of NO₂ ^– on methane oxidation. MDD occurred in media containing only NH₄ ⁺, although the total nitrogen removal efficiency was very low. These results show that NO₂ ^– and NH₄ ⁺, in the presence of NO_x ^–, do not inhibit but rather promote MDD. Consequently, NH₄ ⁺ does not need to be completely oxidized to NO₃ ^– in the nitrification reactor before MDD. However, under psychrophilic conditions, NO₂ ^– seriously inhibited MDD. Therefore, the nitrification reactor must not discharge effluent containing NO₂ ^– under psychrophilic conditions.

Adaptation of anaerobic ammonium-oxidising consortium to synthetic coke-ovens wastewater by S. Toh; N. Ashbolt (pp. 344-352).

A consortium with autotrophic anaerobic ammonium oxidising (AAAO) activity was developed from municipal sludge, and its ability to remove high ammonium concentrations in a toxic wastewater such as coke ovens wastewater is presented here. The enriched AAAO consortium was acclimatised to a synthetic coke ovens wastewater to establish anaerobic ammonium oxidation (AAO) activity. Phenol was the main carbon component of the synthetic wastewater whereby it was added stepwise from 50±10 to 550±10 mg l^–1 into an anammox enrichment medium. Ammonium-N removal was initially impaired; however, it gradually recovered. After 15 months of further selection and enrichment, the ammonium removal rate reached 62±2 mg NH₄ ⁺-N l^–1 day^–1, i.e. 1.5 times the rate in the original AAAO reactor. The new consortium demonstrated higher ammonium and nitrite removal rates, even under phenol perturbation (up to 330±10 mg l^–1). It is therefore concluded that the AAO activity in the consortium was resistant to high phenol and has potential for treating coke-ovens wastewater.

Evaluation of white-rot fungi for detoxification and decolorization of effluents from the green olive debittering process by G. Aggelis; C. Ehaliotis; F. Nerud; I. Stoychev; G. Lyberatos; G. Zervakis (pp. 353-360).

Wastewater produced by the debittering process of green olives (GOW) is rich in polyphenolics and presents high chemical oxygen demand and alkalinity values. Eight white-rot fungi (Abortiporus biennis, Dichomitus squalens, Inonotus hispidus, Irpex lacteus, Lentinus tigrinus, Panellus stipticus, Pleurotus ostreatus and Trametes hirsuta) were grown in GOW for 1 month and the reduction in total phenolics, the decolorization activity and the related enzyme activities were compared. Phenolics were efficiently reduced by P. ostreatus (52%) and A. biennis (55%), followed by P. stipticus (42%) and D. squalens (36%), but only P. ostreatus had high decolorization efficiency (49%). Laccase activity was the highest in all of the fungi, followed by manganese-independent peroxidase (MnIP). Substantial manganese peroxidase (MnP) activity was observed only in GOW treated with P. ostreatus and A. biennis, whereas lignin peroxidase (LiP) and veratryl alcohol oxidase (VAOx) activities were not detected. Early measurements of laccase activity were highly correlated (r ²=0.91) with the final reduction of total phenolics and could serve as an early indicator of the potential of white-rot fungi to efficiently reduce the amount of total phenolics in GOW. The presence of MnP was, however, required to achieve efficient decolorization. Phytotoxicity of GOW treated with a selected P. ostreatus strain did not decline despite large reductions of the phenolic content (76%). Similarly, in GOW treated with purified laccase from Polyporus pensitius, a reduction in total phenolics which exceeded 50% was achieved; however, it was not accompanied by a decline in phytotoxicity. These results are probably related to the formation of phenoxy radicals and quinonoids, which re-polymerize in the absence of VAOx but do not lead to polymer precipitation in the treated GOW.

Effects of 2,4-dichlorophenol on activated sludge by E. Sahinkaya; F. Dilek (pp. 361-367).

The effects of 2,4-dichlorophenol (2,4-DCP) on both acclimated and unacclimated activated sludge were investigated in batch reactors. The IC₅₀ values on the basis of maximum specific growth rate (µ _m), percent chemical oxygen demand (COD) removal efficiency and sludge activity were found to be 72, 60 and 47 mg l^–1, respectively, for unacclimated culture. The percent COD removal efficiencies of unacclimated culture were affected adversely, even at low concentrations, whereas culture acclimated to 75 mg 2,4-DCP l^–1 could tolerate about 200 mg 2,4-DCP l^–1on the basis of COD removal efficiency. Although yield coefficient values of unacclimated culture increased surprisingly to very high values with the addition of 2,4-DCP, a linear decrease with respect to 2,4-DCP concentrations was observed for acclimated culture. Although no removal was observed with unacclimated culture, almost complete removal of 2,4-DCP up to a concentration of 148.7 mg l^–1 was observed with acclimated culture. It was showed that the culture could use 2,4-DCP as sole organic carbon source, although higher removal efficiencies in the presence of a readily degradable substrate were observed. Culture acclimated to 4-chlorophenol used 2,4-DCP as sole organic carbon source better than those acclimated to 2,4-DCP.

Use of a two-phase partitioning bioreactor for degrading polycyclic aromatic hydrocarbons by a Sphingomonas sp. by T. Janikowski; D. Velicogna; M. Punt; A. Daugulis (pp. 368-376).

A two-phase partitioning bioreactor (TPPB) utilizing the bacterium Sphingomonas aromaticivorans B0695 was used to degrade four low molecular weight (LMW) polycyclic aromatic hydrocarbons (PAHs). The TPPB concept is based on the use of a biocompatible, immiscible organic solvent in which high concentrations of recalcitrant substrates are dissolved. These substances partition into the cell-containing aqueous phase at rates determined by the metabolic activity of the cells. Experiments showed that the selected solvent, dodecane, could be successfully used in both solvent extraction experiments (to remove PAHs from soil) and in a TPPB application. Further testing demonstrated that solvent extraction from spiked soil was enhanced when a solvent combination (dodecane and ethanol) was used, and it was shown that the co-solvent did not significantly affect TPPB performance. The TPPB achieved complete biodegradation of naphthalene, phenanthrene, acenaphthene and anthracene at a volumetric consumption rate of 90 mg l^–1 h^–1 in approximately 30 h. Additionally, a total of 20.0 g of LMW PAHs (naphthalene and phenanthrene) were biodegraded at an overall volumetric rate of 98 mg l^–1 h^–1 in less than 75 h. Degradation rates achieved using the TPPB and S. aromaticivorans B0695 are much greater than any others previously reported for an ex situ PAH biodegradation system operating with a single species.

Detection and characterization of erythromycin-resistant methylase genes in Gram-positive bacteria isolated from poultry litter by A. Khan; M. Nawaz; S. Khan; R. Steele (pp. 377-381).

The epidemiology of four erythromycin-resistant methylase (erm) genes, ermA, ermB, ermC and msrA, was determined in erythromycin-resistant staphylococci, enterococci and streptococci isolated from poultry litter. All isolates were resistant to multiple antibiotics. Southern hybridization indicated that 4 of the 20 staphylococci contained the ermC gene on plasmids: on a 2.2 kb plasmid in Staphylococcus hominis and S. sciuri, on a 6.0 kb plasmid in S. xylosus, and on a 7.0 kb plasmid in S. lentus. In 16 of the 20 staphylococci, the ermA gene was harbored exclusively on the chromosome, as a double chromosomal insert on 8.0 and 6.2 kb EcoRI fragments. None of the staphylococci harbored the msrA gene. Dot-blot analysis indicated that all enterococci and streptococci hybridized with a biotinylated ermB gene probe. Southern hybridization indicated that only 2 of the 19 erythromycin-resistant enterococci contained the ermB gene on plasmids. The gene was localized on 4.0 kb and 5.9 kb plasmids, respectively, in two Enterococcus faecium isolates. Results from our studies indicate that the patterns of occurrence of erm genes, the sizes of the plasmids and the copy numbers of the inserts were different from the existing information on the presence of erm genes in clinical strains of Staphylococcus spp.

Nickel accumulation and nickel oxalate precipitation by Aspergillus niger by A. Magyarosy; R. Laidlaw; R. Kilaas; C. Echer; D. Clark; J. Keasling (pp. 382-388).

A strain of Aspergillus niger isolated from a metal-contaminated soil was able to grow in the presence of cadmium, chromium, cobalt, copper, and unusually high levels of nickel on solid (8.0 mM) and in liquid (6.5 mM) media. This fungus removed >98% of the nickel from liquid medium after 100 h of growth but did not remove the other metals, as determined by inductively coupled plasma spectroscopy. Experiments with non-growing, live fungal biomass showed that nickel removal was not due to biosorption alone, as little nickel was bound to the biomass at the pH values tested. Furthermore, when the protonophore carbonyl cyanide p-(trifluoremetoxy) phenyl hydrazone (FCCP) was added to the actively growing fungus nickel removal was inhibited, supporting the hypothesis that energy metabolism is essential for metal removal. Analytical electron microscopy of thin-sectioned fungal biomass revealed that metal removed from the broth was localized in the form of small rectangular crystals associated with the cell walls and also inside the cell. X-ray and electron diffraction analysis showed that these crystals were nickel oxalate dihydrate.

Sections

Personal tools

Applied Microbiology and Biotechnology (v.59, #2-3)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.59, #2-3)