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Applied Microbiology and Biotechnology (v.57, #1-2)

Integrated production of biodegradable plastic, sugar and ethanol by R. Nonato; P. Mantelatto; C. Rossell (pp. 1-5).

Poly 3-hydroxybutyric acid (PHB) and related copolymers can be advantageously produced when integrated into a sugarcane mill. In this favorable scenario, the energy necessary for the production process is provided by biomass. Carbon dioxide emissions to the environment are photosynthetically assimilated by the sugarcane crop and wastes are recycled to the cane fields. The polymer can be produced at low cost considering the availability of a low-price carbon source and energy.

Microbial production of poly-D-3-hydroxybutyrate from CO₂ by A. Ishizaki; K. Tanaka; N. Taga (pp. 6-12).

This short review covers the biotechnological aspects of the production of poly-D-3-hydroxybutyric acid, P(3HB), from H₂, O₂ and CO₂ by autotrophic culture of the hydrogen-oxidizing bacterium, Ralstonia eutropha. Considering the efficiency of utilization of a gas mixture as substrate, a practical fermentation process using R. eutropha for the mass production of P(3HB) from CO₂ should be designed on the basis of a recycled-gas, closed-circuit culture system. Also, maintaining the O₂ concentration in a gas phase lower than 6.9% (v/v) is essential to prevent the gas mixture from exploding. Our study, using an explosion-proof fermentation bench plant and a two-stage culture system with a newly designed air-lift fermenter, demonstrated that very high P(3HB) yield and productivity could be obtained while the O₂ concentration was maintained below 6.9%. However, a study on the continuous production of P(3HB) from CO₂ by chemostat culture of R. eutropha revealed that the productivity and content of P(3HB) in the cells was considerably lower than by fed-batch culture. It is deduced that the use of the hydrogen-oxidizing bacterium, Alcaligenes latus, which accumulates P(3HB) even in the exponential growth phase, will be useful for the effective production of P(3HB) from CO₂.

Taxol: biosynthesis, molecular genetics, and biotechnological applications by S. Jennewein; R. Croteau (pp. 13-19).

Over the past decade, Taxol and its closely related structural analogue Taxotere have emerged as very important antitumor agents. Their widespread use in the treatment of a variety of cancer types, their likely approval for the treatment of additional forms of cancer, and their use at earlier stages of intervention will lead to increased demand for these drugs in the future. Because of yield considerations, Taxol and Taxotere are currently derived via semisynthesis from the advanced taxoid 10-deacetylbaccatin III, which must be isolated from yew (Taxus) trees. Thus, efforts are underway to produce Taxol (and other advanced taxoids for use in semisynthesis) by alternate, biotechnological means. This article provides a current overview of research on taxoid biosynthesis and an assessment of bioengineering applications for taxoid production in yew cell culture.

Feasibility of bioremediation by white-rot fungi by S. Pointing (pp. 20-33).

The ligninolytic enzymes of white-rot fungi have a broad substrate specificity and have been implicated in the transformation and mineralization of organopollutants with structural similarities to lignin. This review presents evidence for the involvement of these enzymes in white-rot fungal degradation of munitions waste, pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, bleach plant effluent, synthetic dyes, synthetic polymers, and wood preservatives. Factors relating to the feasibility of using white-rot fungi in bioremediation treatments for organopollutants are discussed.

Influence of the fluidity of the membrane on the response of microorganisms to environmental stresses by L. Beney; P. Gervais (pp. 34-42).

The aim of this mini-review is to relate membrane physical properties to the adaptation and resistance of microorganisms to environmental stresses. In the first part, the effects of various stresses on the structure and dynamic properties of phospholipid and biological membranes are presented. The compensation of these effects, i.e., change in membrane fluidity, phase transitions, by the active cellular control of the membrane chemical composition, is then described. In this natural process, the change in membrane fluidity is viewed as the detecting "input" signal that initiates the regulation, activating proteic effectors that in turn may influence the chemical composition of the membrane (feedback). This adaptation system allows the maintenance of the physical characteristics of membranes and, thereby, of their functionality. When environmental stresses are extreme and occur abruptly, the regulation process may not compensate for the changes in the membrane physical characteristics. In such cases, important variations in the membrane fluidity and structure may induce cellular damages and cell death. However, the lethal consequences are not systematically observed because protective effects of changes in the membrane physical state on the resistance to stresses are also reported.

Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens by C. Hartley; F. Manford; S. Burton; R. Dorrington (pp. 43-49).

While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli. This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions. Hydantoinase and N-carbamoylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in complete medium, enzyme activity can be detected only in early stationary growth phase. In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitrogen source and the presence of inducer, 2-thiouracil, in the growth medium. Growth with (NH₄)₂SO₄ as the nitrogen source repressed the production of both enzymes (nitrogen repression) and also resulted in a rapid, but reversible loss of hydantoinase activity in induced cells (ammonia shock). Mutant strains with inducer-independent production of the enzymes and/or altered response to nitrogen control were isolated. Of greatest importance for industrial application was strain RU-ORPN1F9, in which hydantoinase and NCAAH enzyme activity was inducer-independent and no longer sensitive to nitrogen repression or ammonia shock. Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterologous expression systems.

Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by G. Chen; G. Zhang; S. Park; S. Lee (pp. 50-55).

Large scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by Aeromonas hydrophila 4AK4 was examined in a 20,000 l fermentor. Cells were first grown using glucose as a carbon source, and polyhydroxyalkanoate (PHA) biosynthesis was triggered by the addition of lauric acid under conditions of limited nitrogen or phosphorus. When cells first grown in a medium containing 50 g glucose l^–1 were further cultivated after the addition of 50 g lauric acid l^–1 under phosphorus limitation, a final cell concentration, PHA concentration and PHA content of 50 g l^–1, 25 g l^–1, and 50 wt%, respectively, were obtained in 46 h, equivalent to PHA productivity of 0.54 g l^–1 h^–1. The copolymer produced was found to be a random copolymer, and the 3HHx fraction was 11 mol%.

Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate by C-C. Chen; C-Y. Lin; J-S. Chang (pp. 56-64).

In this study, local sewage sludge was acclimated to establish H₂-producing enrichment cultures, which were used to convert sucrose to H₂with continuously stirred anaerobic bioreactors. The steady-state behaviors of cell growth, substrate utilization, and product formation were closely monitored. Kinetic models were developed to describe and predict the experimental results from the H₂-producing cultures. Operation at dilution rates (D) of 0.075–0.167 h^–1 was preferable for H₂ production, resulting in a H₂ concentration of nearly 0.02 mol/l. The optimal hydrogen production rate was 0.105 mol/h occurring at D=0.125 h^–1. The major volatile fatty acid produced was butyric acid (HBu), while acetic acid and propionic acid were also produced in lesser quantities. The major solvent product was ethanol, whose concentration was only 15% of that of HBu, indicating that the metabolic flow favors H₂ production. The proposed model was able to interpret the trends of the experimental data. The maximum specific growth rate (µ _max), Monod constant (K s), and yield coefficient for cell growth (Y x/s) were estimated as 0.172 h^–1, 68 mg COD/l, and 0.1 g/g, respectively. The model study also suggests that product formation in the continuous hydrogen-producing cultures was essentially a linear function of biomass concentration.

Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor by Y. Ueno; S. Haruta; M. Ishii; Y. Igarashi (pp. 65-73).

Changes in product formation during carbohydrate fermentation by anaerobic microflora in a continuous flow stirred tank reactor were investigated with respect to the dilution rate in the reactor. In the fermentation by methanogenic microflora, stable methane fermentation, producing methane and carbon dioxide, was observed at relatively low dilution rates (less than 0.33 d^–1 on glucose and 0.20 d^–1 on cellulose). Decomposition of cellulose in the medium was a rate-limiting step in the reaction, because glucose was easily consumed at all applied dilution rates (0.07–4.81 d^–1). Intermediate metabolites of methane fermentation, such as lactate, ethanol, acetate, butyrate, formate, hydrogen, and carbon dioxide, were accumulated as dilution rate increased. Maximum yield of hydrogen was obtained at 4.81 d^–1 of dilution rate (0.1 mol/mol glucose on glucose or 0.7 mol/mol hexose on cellulose). Lactate was the major product on glucose (1.2 mol/mol glucose), whereas ethanol was predominant on cellulose (0.7 mol/mol hexose). An analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S rDNA of the microflora indicated that changes in the microbial community took place at various dilution rates, and these changes appeared to correspond to the changes in product distributions. Sequence analyses of the DGGE fragments revealed the probable major population of the microflora. A band closely related to the microorganisms of thermophilic anaerobic bacteria was detected with strong intensity on both glucose and cellulose. Differences in the production yield of hydrogen could have been caused by different populations of microorganisms in each microflora. In the case of cellulose, increasing the dilution rate brought about an accumulation of microorganisms related to Clostridia species that have cellulolytic activity, this being in accordance with the notion of cellulose decomposition being the rate-limiting reaction.

Effects of nutrient supplements on biological efficiency, quality and crop cycle time of maitake (Grifola frondosa) by Q. Shen; D. Royse (pp. 74-78).

The effects of various combinations of wheat bran, rye and millet (at 20% and 30% of total dry substrate wt) on crop cycle time, biological efficiency (BE) and mushroom quality were evaluated for a commercially used isolate of Grifola frondosa (maitake). Supplements were combined with a basal ingredient of mixed oak (primarily red oak) sawdust, and the resulting mixture was pasteurized, cooled, inoculated and bagged with an autoclaving mixer. Times to mushroom primordial formation and mushroom harvest were recorded, and mushroom quality was rated on a scale of 1–4, where 1 was the highest quality and 4 was the lowest quality. The combinations of 10% wheat bran, 10% millet and 10% rye (BE 47.1%, quality 1.8 and crop cycle 12 weeks) and 10% wheat bran plus 20% rye (BE 44%, quality 1.7 and crop cycle 10 weeks) gave the most consistent yields and best basidiome quality over time.

Optimization of stereoselective ketone reduction by the white-rot fungus Merulius tremellosus ono991 by A. Hage; H. Schoemaker; J. Field (pp. 79-84).

A recently isolated white-rot fungal strain, Merulius tremellosus ono991, displays high stereoselectivity during the reduction of arylketones. In order to increase the productivity and specific yield of the optically active alcohols, the culture conditions for the reduction of the model ketone compound 1′-acetonaphtone to α-methyl-1-naphtalenemethanol were optimized with respect to oxygen supply, choice of primary substrate and arylketone concentration. Alternative electron acceptors were also used to elucidate the role of reduction equivalents in the reduction process. The optimal yields of α-methyl-1-naphtalenemethanol were obtained in N₂-flushed incubations with glycerol as primary substrate. The specific yield was increased from 57% to 98% compared to incubations under air with glucose. Most of the yield increase was due to N₂-flushing and could be attributed to two factors. First, an increased stability of the product, α-methyl-1-naphtalenemethanol, in anaerobic compared to aerobic atmosphere was demonstrated. Second, fermentative metabolism increased reduced enzyme cofactors available for the reduction. Diverting reducing equivalents away from fermentation with alternative electron acceptors correlated with a decreased yield of α-methyl-1-naphtalenemethanol. Furthermore, the dependency of ketone reductase for common occurring metabolic reducing equivalents, NAD(P)H, was demonstrated by the reduction of 1′-acetonaphtone in cell extracts of M. tremellosus ono991.

Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase by A. Leontievsky; N. Myasoedova; B. Baskunov; L. Golovleva; C. Bucke; C. Evans (pp. 85-91).

Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4°C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the unfavourable effects of alkaline pH, high temperature and the action of inhibitors. A combination of these properties of immobilized laccase resulted in the ability to oxidize 2,4,6-trichlorophenol (2,4,6-TCP) at 50°C at pH 7.0. The reactions of soluble and immobilized laccase with 2,4,6-TCP were examined in the presence and absence of redox mediators. 3,5-Dichlorocatechol, 2,6-dichloro-1,4-benzoquinone and 2,6-dichloro-1,4-hydroquinone were found to be the primary products of 2,4,6-TCP oxidation by laccase; oligo- and polymeric compounds were also found.

The use of extracellular enzymes from Streptomyces albus ATCC 3005 for the bleaching of eucalyptus kraft pulp by V. Antonopoulos; M. Hernandez; M. Arias; E. Mavrakos; A. Ball (pp. 92-97).

The suitability of culture supernatant from Streptomyces albus ATCC 3005 for use in the biobleaching of eucalyptus kraft pulp was investigated. S. albus was found to grow on a minimal salts medium containing oat spelts xylan and yeast extract as the main carbon and nitrogen sources, respectively. Maximal extracellular xylanase and peroxidase production was detected after 120 h (11.97 U ml^–1) and 72 h (0.58 U ml^–1), respectively. Importantly, no cellulase activity could be detected. When the effect of pH on enzyme activity was examined, maximal xylanase and peroxidase activity was obtained at pH 6.5 and pH 9.9, respectively. The optimum hydrogen peroxide (H₂O₂) concentration for peroxidase activity was found to occur at 20 mM, with peroxidase remaining active at 100 mM H₂O₂ after 1 h incubation at 53°C; the half-life of the enzyme at that temperature was estimated to be 33 min. Short-term (1 h) biobleaching of eucalyptus kraft pulp with culture supernatant from S. albus in the presence of H₂O₂ resulted in a significant reduction of kappa number (2.85 units) with no change in viscosity. These results suggest a potential application of cellulase-free culture supernatants from S. albus in biobleaching.

Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum by E.-M. Ko; Y.-E. Leem; H. Choi (pp. 98-102).

Ganoderma lucidum, a medicinal white-rot basidiomycete, produces many laccase isozymes in liquid culture. Three laccase isozymes (GaLc 1, 2, 3) have been purified 32.4-fold from the crude enzyme protein through anion exchange chromatography, preparative gel electrophoresis, and electroelution. Their estimated molecular weights are 65–68 kDa, and they contain 7–10% N-linked carbohydrates. The three isozymes have identical N-terminal amino acid sequences: G-I-G-P-T. The optimum pH and temperature both for each isozyme singly and the isozyme mixture are pH 3.5 and 20°C, respectively. One isozyme (GaLc 3) is quite stable at pH 4.0–10.0, and shows good stability when incubated at temperatures lower than 40°C. The K _m values of GaLc 3 for o-tolidine and 2,2'-azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) are 401.6 µM and 3.7 µM respectively, and the V _max of GaLc 3 for these substrates is 0.0198 OD min^–1unit^–1 and 0.0142 OD min^–1unit^–1, respectively.

A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1 by M. Okamoto; Y. Yonejima; Y. Tsujimoto; Y. Suzuki; K. Watanabe (pp. 103-108).

A collagenolytic protease was purified to homogeneity from thermophilic Bacillus sp. strain MO-1. The protease from strain MO-1 showed high activity toward type I and IV collagens and gelatin. However, peptide substrates (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg and 2-furylacryloyl-Leu-Gly-Pro-Ala) for collagenases were inert as substrates. The collagenolytic protease cleaved oxidized insulin B-chain at 11 sites and degraded type I and IV collagens into anonymous small pieces, suggesting that the protease digests collagens at multiple sites. The collagenolytic protease was far more thermostable than a mesophilic Clostridium histolyticum collagenase. The collagenolytic protease possesses two salient features: (1) it has a very large molecular mass, 210 kDa, and consists of two, identical 105-kDa subunits; (2) it belongs to a serine protease group. The high molecular mass is unique among serine proteases but common for collagenases. The features of the enzyme from strain MO-1 suggest that it is a new collagenolytic protease which is distinct from previously reported collagenases and serine proteases.

A novel alkaline endoglucanase from an alkaliphilic Bacillus isolate: enzymatic properties, and nucleotide and deduced amino acid sequences by K. Endo; Y. Hakamada; S. Takizawa; H. Kubota; N. Sumitomo; T. Kobayashi; S. Ito (pp. 109-116).

A highly alkaline endo-1,4-β-glucanase (Egl) was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM-N252. The optimal pH for activity was as high as 10, and the optimal temperature was 55°C. The molecular mass and isoelectric point were around 50 kDa and pH 4.2, respectively. The enzyme hydrolyzed carboxymethyl cellulose in a random fashion. Unlike previously reported Egls, the enzyme was highly active on p-nitrophenyl cello-oligosaccharides and acid-swollen cellulose, and its activity was stimulated by cellobiose at high concentrations. The entire gene for the enzyme contained a 1,476-bp single open reading frame encoding 492 amino acids, including a 29-amino-acid signal peptide. The mature enzyme (463 amino acids: 51,174 Da) exhibited moderate homology to other family 5 alkaline Egls. In the C-terminal region, a carbohydrate-binding module that belongs to family XII was repeated. Furthermore, four and six repeats of Pro-Pro-Ser/Thr-Glu/Asp-Pro-(Glu) were found immediately before the first and second carbohydrate-binding modules, respectively.

Purification and properties of a β-1,6-glucanase from Streptomyces sp. EF-14, an actinomycete antagonistic to Phytophthora spp. by K. Fayad; A.-M. Simao-Beaunoir; A. Gauthier; C. Leclerc; H. Mamady; C. Beaulieu; R. Brzezinski (pp. 117-123).

Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A β-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-β-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of β-1,6-glucanase activity always preceded that of β-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize β-1,6-links flanked by β-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this β-1,6-glucanase is a non-lytic enzyme. This is the first β-1,6-glucanase characterized from an actinomycete.

Overproduction, purification, and characterization of the Trichoderma reesei hydrophobin HFBI by S. Askolin; T. Nakari-Setälä; M. Tenkanen (pp. 124-130).

Many characteristics of fungal hydrophobins, such as an ability to change hydrophobicity of different surfaces, have potential for several applications. The large-scale processes of production and isolation of these proteins susceptible to aggregation and attachment to interfacial surfaces still needs to be studied. We report for the first time on a method for a gram-scale production and purification of a hydrophobin, HFBI of Trichoderma reesei. A high production level of the class II hydrophobin (0.6 g l^–1) was obtained by constructing a T. reesei HFBI-overproducing strain containing three copies of the hfb1 gene. The strain was cultivated on glucose-containing medium, which induces expression of hfb1. HFBI hydrophobin was purified from the cell walls of the fungus because most of the HFBI was cell-bound (80%). Purification was carried out with a simple three-step method involving extraction of the mycelium with 1% SDS at pH 9.0, followed by KCl precipitation to remove SDS, and hydrophobic interaction chromatography. The yield was 1.8 g HFBI from mycelium (419 g dw), derived from 15 l of culture. HFBI was shown to be rather unstable to N-terminal asparagine deamidation and also, to some extent, to non-specific proteases although its thermostability was excellent.

Establishment of a hyper-protein production system in submerged Aspergillus oryzae culture under tyrosinase-encoding gene (melO) promoter control by H. Ishida; K. Matsumura; Y. Hata; A. Kawato; K. Suginami; Y. Abe; S. Imayasu; E. Ichishima (pp. 131-137).

UV-mediated mutagenesis generated a high glucoamylase-producing mutant of Aspergillus oryzae exhibiting strong melanization in solid-state culture. Expression of the glucoamylase-encoding gene (glaB), which is specifically expressed in solid-state culture, and the tyrosinase-encoding gene (melO), was analyzed using an E. coli β-glucuronidase (GUS) reporter assay to investigate this phenomenon. Although no common regulation was found for melO and glaB expression, the former was greatly enhanced in submerged culture. Interestingly, the melO promoter was about four times stronger for GUS production than the powerful promoters amyB, glaA, and modified agdA, previously isolated for industrial heterologous gene expression in A. oryzae. These findings indicated that the melO promoter would be suitable for hyper-production of heterologous protein in Aspergillus. The glaB-type glucoamylase selected as the target protein was produced in a submerged culture of A. oryzae under the control of the melO promoter. The maximum yield was 0.8 g/l broth, and the total extracellular protein purity was 99%. Repeated batch culture, to improve productivity, gave a maximum yield of 3.3 g/l broth. The importance of this work is in the establishment of a both high-level and high-purity protein overproduction system in A. oryzae by use of the melO promoter.

Cloning of the cel9A gene and characterization of its gene product from marine bacterium Pseudomonas sp. SK38 by S. Ryu; S. Cho; S. Park; W. Lim; M. Kim; S. Hong; D. Bae; Y. Park; B. Kim; H. Kim; H. Yun (pp. 138-145).

The yellow-pigmented bacterial strain causing green spot rot and death of laver was isolated from Porphyra dentata. This strain has been identified as Pseudomonas sp., harboring agarase, xylanase, and protease activity, as well as carboxymethyl-cellulase (CMCase). Using genomic DNA from the Pseudomonas sp. SK38 digested with Sau3AI and ligated into pBluescript II KS+, we isolated a cel gene encoding a CMCase in Pseudomonas sp. SK38. A 4.5-kb fragment was subcloned into pKR400. The structure of the cel9A gene consists of an open reading frame of 1,521 bp starting with a GTG start codon and ending with a TAG stop codon. It thus encodes 506 amino acid residues of a protein with a calculated molecular weight of 52,636 daltons plus a signal peptide of 22 amino acids. The deduced amino acid sequence of the cel9A protein is similar to the same protein of Clostridium thermocellum. It contains, in particular, the two conserved regions of the glycoside hydrolase family 9. The apparent molecular mass of the Cel9A protein is 52 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is most active at pH 6–7 and an optimal temperature of around 30°C.

Cloning, sequence analysis and expression of a gene encoding an organic solvent- and detergent-tolerant cholesterol oxidase of Burkholderia cepacia strain ST-200 by N. Doukyu; R. Aono (pp. 146-152).

Burkholderia cepacia strain ST-200 produces an extracellular cholesterol oxidase which is stable and highly active in the presence of organic solvents. This cholesterol oxidase produces 6β-hydroperoxycholest-4-en-3-one from cholesterol, with the consumption of two moles of O₂ and the formation of one mole of H₂O₂. The structural gene encoding the cholesterol oxidase was cloned and sequenced. The primary translation product was predicted to be 582 amino acid residues. The mature product is composed of 539 amino acid residues and is preceded by a signal sequence of 43 residues. The cloned gene was expressed as an active product in Escherichia coli and the product was localized in the periplasmic space. The cholesterol oxidase produced from E. coli was purified to homogeneity from the periplasmic fraction. The purified enzyme was highly stable in the presence of various organic solvents or detergents, as compared with the commercially available cholesterol oxidases tested.

Fermentation of starch for enhanced alkaline protease production by constructing an alkalophilic Bacillus pumilus strain by Y. Feng; W. Yang; S. Ong; J. Hu; W. Ng (pp. 153-160).

A new engineering strain, Bacillus pumilus c172-14 (pBX 96), was obtained by introducing the pBX 96 plasmid, which carries the α-amylase amy gene, into the host strain of alkalophilic Bacillus pumilus c172 via transformation. The newly constructed strain was found to express the amy gene and could use starch instead of glucose or starch hydrolysate as carbon source for its fermentation of alkaline protease. The pBX 96 plasmid in the new host was found to be segregationally and structurally stable. The expression of amy gene did not affect the host strain's resistance to bacteriophages. Moreover, the level of alkaline protease was improved significantly compared with the parent strain. The constructed strain gave a maximum alkaline protease activity of 14,014 U/ml in shaking flask after 48 h cultivation when growing in a medium containing 6% corn meal, 4% soybean flour, 0.4% Na₂HPO₄, 0.03% KH₂PO₄, 0.02% MgCl₂, 0.3% CaCl₂, 0.25% Na₂CO₃, 0.1% glucose, and 20 µg/ml kanamycin (pH 7.0). The optimal pH value and temperature of the alkaline protease were 11.0 and 40°C, respectively. This enzyme was stable over a pH range of 8–11. Its residual activity remained at 100% when treated under a temperature of less than 45°C for 30 min. The corresponding residual activity reduced to 65% of its optimal value at 60°C for 30 min. The alkaline protease was a kind of serine protease, which was demonstrated by the complete inactivation by PMSF (1 mM). This newly constructed strain will be useful in the alkaline protease industry.

Suitability of Anabaena PCC7120 expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis for biotechnological application by A. Lluisma; N. Karmacharya; A. Zarka; E. Ben-Dov; A. Zaritsky; S. Boussiba (pp. 161-166).

We present evidence that Anabaena PCC7120 (A.7120) strains expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis (Bti) have a strong potential for biotechnological application. Characterization of two 4-year-old recombinant A.7120 clones constructed previously in our laboratory [clone 7 and clone 11, each carrying three Bti genes (cry4Aa, cry11Aa, and p20)] revealed three facts. First, the Bti genes were stable in A.7120 even in the absence of antibiotic selection when the genes were integrated in the chromosome (in clone 11); and the genes were also stable as plasmid-borne constructs (in clone 7), provided the cultures were maintained under continued selection. Second, clone 7 (kept under selection) and clone 11 (either kept or not kept under selection) continued to be mosquitocidal through 4 years of culture. Third, growth of the recombinant clones was comparable to the wild type under optimal growth conditions, indicating that growth was not compromised by the expression of toxin genes. These results clear the way for the development of mass production techniques for A.7120 strains expressing Bti toxin genes.

Effect of overexpression of Saccharomyces cerevisiae Pad1p on the resistance to phenylacrylic acids and lignocellulose hydrolysates under aerobic and oxygen-limited conditions by S. Larsson; N.-O. Nilvebrant; L. Jönsson (pp. 167-174).

Lignocellulose hydrolysates, obtained by acid hydrolysis for production of bioethanol, contain, in addition to fermentable sugars, compounds that inhibit the fermenting micro-organism. One approach to alleviate the inhibition problem is to use genetic engineering to introduce increased tolerance. Phenylacrylic acid decarboxylase (Pad1p) catalyses a decarboxylation step, by which aromatic carboxylic acids are converted to the corresponding vinyl derivatives. Pad1p-overexpressing Saccharomyces cerevisiae was cultivated in synthetic medium in the presence of model compounds, ferulic acid [(2 E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoic acid] and cinnamic acid [(2 E)-3-phenylprop-2-enoic acid], as well as in a dilute acid hydrolysate of spruce to examine the resistance against fermentation inhibitors. Overexpression of S. cerevisiae phenylacrylic acid decarboxylase (Pad1p) resulted in an improved growth rate and ethanol productivity in the presence of ferulic acid, cinnamic acid, and in a dilute acid hydrolysate of spruce. Vinyl guaiacol (2-methoxy-4-vinylphenol) was identified as a major metabolite of ferulic acid, and dihydroferulic acid [3-(4-hydroxy-3-methoxyphenyl)propanoic acid] was detected under oxygen-limited conditions. Styrene (vinylbenzene) and dihydrocinnamic acid (3-phenylpropanoic acid) were identified as metabolites of cinnamic acid. Transformants overexpressing Pad1p had the ability to convert ferulic and cinnamic acid at a faster rate than a control transformant (PAD^C) not overexpressing Pad1p. This enabled faster growth for Pad1p-overexpressing transformants under both aerobic and oxygen-limited conditions. Pad1p activity was also studied using non-growing cells. The overexpressing transformants showed approximately tenfold higher activity than PAD^C. The Pad1p overexpressing transformants also showed a 22–25% faster glucose consumption rate, a 40–45% faster mannose consumption rate, and a 24–29% faster ethanol production rate in the dilute acid hydrolysate of spruce.

Coflocculation of Escherichia coli and Schizosaccharomyces pombe by X. Peng; J. Sun; C. Michiels; D. Iserentant; H. Verachtert (pp. 175-181).

Several yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, Saccharomyces cerevisiae and Schizosaccharomyces pombe, were found to coaggregate with Escherichia coli, but S. pombe showed much less coflocculation than the other yeasts (Peng et al. 2001) ). S. pombe is known to have galactose-rich cell walls and we investigated whether this might be responsible for its different behavior by studying the wild-type TP4-1D, with a mannose to galactose ratio of 1 to 1.2, and the glycosylation mutant gms1Δ (Man:Gal=1:0). The wild-type induced very low levels of coflocculation (3%) while gms1Δ induced a remarkable amount of coflocculation (48%). Coflocculation of the mutant was inhibited by mannose but not affected by galactose or glucose. The S. cerevisiae mnn2 mutant, with a mannan structure similar to gms1Δ, also showed a high degree of coflocculation (40%). However, S. cerevisiae mutant mnn9, with a mature core similar to S. pombe, showed decreased coflocculation (21.3%). Both these S. cerevisae mutants were sensitive to mannose inhibition. Coflocculation of E. coli and gms1Δ also could be inhibited by gms1Δ mannan and plant lectins, such as HHA, GNA and NPA, specific to either α-1-3- or α-1-6-linked mannosyl units. From these results we conclude that the E. coli lectins may have specificity for α-1-6- and α-1-3-linked mannose residues either in the outer chain or in the core of S. pombe, but in wild-type strains these mannose residues are shielded by galactose residues.

Strains degrading polysaccharides produced by bacteria from paper machines by M. Rättö; A. Mustranta; M. Siika-aho (pp. 182-185).

Biofilm-degrading enzymes are potential agents for slime control in paper machines. In this work, extracellular polysaccharides were produced by bacteria isolated from paper machines and the isolated polysaccharides were used as substrates for the screening of polysaccharide-degrading microbes. Polysaccharide yields of 1.5–3.5 g/l were obtained by ethanol precipitation from cultures of strains of Klebsiella pneumoniae, Bacillus licheniformis and Pseudomonas fluorescens on sucrose medium. Two K. pneumoniae strains apparently produced an identical heteropolysaccharide containing galacturonic acid. Fructose-containing polysaccharides were the main products of B. licheniformis and P. fluorescens. Bacteria capable of hydrolyzing the fructose-containing polymers (levans) appeared to be relatively common among the strains selected for screening. None of the bacteria or mixed cultures screened were able to utilize the Klebsiella heteropolysaccharides.

Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system by V. Hernández-Montalvo; F. Valle; F. Bolivar; G. Gosset (pp. 186-191).

Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid and efficient manner. Therefore, the development of mutant strains that avoid this regulatory system is of special interest to fermentation processes. In the present study, the utilization of sugar mixtures by an Escherichia coli mutant strain devoid of the phosphotransferase system (PTS) was characterized. This mutant can transport glucose (PTS^– Glucose⁺ phenotype) by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose- or xylose-utilization in the wild-type strain. However, under the same culture conditions with the PTS^– Glucose⁺ mutant, glucose and arabinose were co-metabolized, but glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild-type strain sequentially utilized glucose, arabinose and finally, xylose. In contrast, the PTS^– Glucose⁺ strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the PTS^– Glucose⁺ strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild-type strain. [¹⁴C]-Xylose uptake experiments showed that in the PTS^– Glucose⁺ strain, galactose permease increases xylose transport capacity and the observed partial repression of xylose utilization depends on the presence of intracellular glucose.

Aflatoxin genes and the aflatoxigenic potential of Koji moulds by P. Broek; A. Pittet; H. Hajjaj (pp. 192-199).

Sixty-four Aspergillus isolates, 54 of which originated from food fermentations, and 18 Aspergillus reference strains were identified and screened for the presence of aflatoxin genes aflR and omt-1. Among the Koji moulds, not only A. oryzae but also A. flavus strains were found. Furthermore, 27% of A. oryzae and 93% of A. flavus strains lacked either aflR or both aflR- and omt-1. A selection of 29 strains was also checked for the presence of pksA and nor-1. This revealed large deletions in the aflatoxin gene cluster of some strains. The hybridisation patterns also suggested a polarity in the deletion events, originating in the vicinity of pksA and extending towards omt-1. Other strains exhibited BamHI restriction fragment length polymorphisms (RFLPs) for either aflR or for aflR and omt-1. All aflR and/or omt-1 deletion strains turned out to be unable to produce aflatoxin. The RFLP-carrying strains either produced only traces of aflatoxin or none at all. In 73% of the A. oryzae strains, no apparent deletions were detected with the aflR and omt-1 probes. Nevertheless, after incubation in aflatoxin-inducing media, no aflatoxin B₁ production could be detected in those A. oryzae strains.

High-pressure adaptation by salt stress in a moderately halophilic bacterium obtained from open seawater by T. Tanaka; J. Burgess; P. Wright (pp. 200-204).

High-pressure adaptation was examined using a moderately halophilic bacterium (Micrococcus roseus), which was isolated from open seawater and capable of growing in 15% w/v NaCl (optimum NaCl concentration: 3% w/v). After treatment at 207 MPa, colony-forming units (CFUs) significantly decreased; however, the loss of integral cells after pressure was only 30% when direct cell count was performed microscopically. In order to investigate the piezotolerance of M. roseus under high pressure without morphological change, the survival of cells was examined under pressure at 138 MPa for 2 h. M. roseus in 3% NaCl was still sensitive to pressure at 138 MPa. However, the cells in the third generations showed remarkably increased pressure resistance, and no significant loss of viability was confirmed. Furthermore, when M. roseus was cultured in 1, 3, 5, 10 and 15% NaCl, the survival ratio proportionally increased at increased NaCl concentration. M. roseus cultured in 15% NaCl was remarkably resistant (94.7% viability) to pressure at 138 MPa, even when suspended in lower concentration of NaCl. This suggests that NaCl concentrations in growth culture affect the piezotolerance of M. roseus and that this species has an ability to adapt to high pressure.

Nitrogen-removal with protease as a method to improve the selective delignification of hemp stemwood by the white-rot fungus Bjerkandera sp. strain BOS55 by J. Dorado; J. Field; G. Almendros; R. Sierra-Alvarez (pp. 205-211).

Certain white-rot fungi cause selective removal of lignin from woody substrates. Selective delignification can potentially be applied to biopulping and upgrading animal feeds. Nitrogen nutrient limitation is known to enhance the selectivity of lignin degradation. The relatively high N-content of annual fiber crops is an important drawback for utilizing white-rot fungi for their selective delignification. In this study, removal of N from hemp stemwood with protease was explored as a means of improving the selectivity of lignin degradation by the white-rot fungus Bjerkandera sp. strain BOS55. Various protease treatments followed by hot-water extraction were found to be suitable in lowering the N-content of hemp stemwood by up to 70%. The removal was significantly higher than with hot-water extraction alone, which caused a 39% N-removal. The selectivity of lignin degradation was compared in protease-treated, hot-water treated, untreated and ammonium-spiked hemp stemwood, providing N levels that were, respectively, 0.32-, 0.61-, 1.0- and 5.0-fold relative to the natural N-content in the substrate. Removal of N by hot-water extraction alone or in combination with protease greatly protected the holocellulose fraction from excessive decay during 10 weeks of solid state fermentation. However, the selectivity of lignin decay was only greatly enhanced (three-fold) by the protease treatment, due mostly to a highly improved lignin degradation at the lowest N-level.

Benzo[b]thiophene desulfurization by Gordonia rubropertinctus strain T08 by T. Matsui; T. Onaka; K. Maruhashi; R. Kurane (pp. 212-215).

A benzothiophene-desulfurizing bacterium which has a novel desulfurization pathway was isolated and identified as Gordonia rubropertinctus strain T08. Gas chromatography/mass spectroscopy analysis of the ethyl acetate extract of the culture broth detected benzothiophene sulfoxide, benzothiophene sulfone, benzo[e][1,2]oxathiin S-oxide (BT-sultine), benzo[e][1,2]oxathiin S,S-dioxide (BT-sultone), o-hydroxystyrene, and 2-coumaranone, but not 2-(2'-hydroxyphenyl)ethan-1-al, which has been reported to be a desulfurized product of mesophilic nocardioforms.

Production of a heterologous proteinase A by Saccharomyces kluyveri by K. Møller; L. Tidemand; J. Winther; L. Olsson; J. Piškur; J. Nielsen (pp. 216-219).

In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a reference, S. cerevisiae CEN.PK 113-5D was transformed with the same plasmid and the two strains were characterised in batch cultivations on glucose. The glucose metabolism was found to be less fermentative in S. kluyveri than in S. cerevisiae. The yield of ethanol on glucose was 0.11 g/g in S. kluyveri, compared to a yield of 0.40 g/g in S. cerevisiae. Overexpression of PEP4 led to the secretion of active proteinase A in both S. kluyveri and S. cerevisiae. The yield of active proteinase A during growth on glucose was found to be 3.6-fold higher in S. kluyveri than in the S. cerevisiae reference strain.

Production of a heterologous proteinase A by Saccharomyces kluyveri by K. Møller; L. Tidemand; J. Winther; L. Olsson; J. Piškur; J. Nielsen (pp. 220-220).

Roles of two white-rot basidiomycete fungi in decolorisation and detoxification of olive mill waste water by M. Kissi; M. Mountadar; O. Assobhei; E. Gargiulo; G. Palmieri; P. Giardina; G. Sannia (pp. 221-226).

A Phanerochaete chrysosporium strain was isolated from Moroccan olive mill waste water (OMW) and its ability to degrade OMW in different culture conditions was investigated and compared to that of Pleurotus ostreatus. The results indicated that Ph. chrysosporium isolate is more efficient than Pl. ostreatus in decolorising and detoxifying OMW in the presence of added nutrients. Ph. chrysosporium is able to remove more than 50% of the colour and phenols from OMW within 6 days of incubation, whereas Pl. ostreatus needs more than 12 days to reach similar results in the same conditions. Many factors affecting the treatment of diluted OMW (20%) by Ph. chrysosporium were studied, including the effects of added nutrients, initial pH, temperature and inoculated biomass. Once the optimisation of 20% OMW biodegradation process had been set up, higher OMW concentrations (50%) were tested. The results show that the fungus is capable of reducing all parameters analysed (colour A₃₉₅, phenol content and chemical oxygen demand) by at least 60%, after only 9 days of growth.

The effects of shear force on the formation, structure and metabolism of aerobic granules by J.-H. Tay; Q.-S. Liu; Y. Liu (pp. 227-233).

The effect of shear force on aerobic granulation was studied in four column-type, sequential aerobic sludge blanket reactors. Hydrodynamic turbulence caused by upflow aeration served as the main shear force in the systems. Results showed that aerobic granulation was closely associated with the strength of shear force. Compact and regular aerobic granules were formed in the reactors with a superficial upflow air velocity higher than 1.2 cm s^–1. However, only typical bioflocs were observed in the reactor with a superficial upflow air velocity of 0.3 cm s^–1 during the whole experimental period. The characteristics of the aerobic granules in terms of settling ability, specific gravity, hydrophobicity, polysaccharide and protein content and specific oxygen utilization rate (SOUR) were examined. It was found that the shear force has a positive effect on the production of polysaccharide, SOUR, hydrophobicity of cell surface and specific gravity of granules. The hydrophobicity of granular sludge is much higher than that of bioflocs. Therefore, it appears that hydrophobicity could induce and further strengthen cell–cell interaction and might be the main force for the initiation of granulation. The shear-stimulated production of polysaccharides favors the formation of a stable granular structure. This research provides experimental evidence to show that shear force plays a crucial role in aerobic granulation and further influences the structure and metabolism of granules.

Contribution of suspended and sorbed groundwater bacteria to degradation of dissolved and sorbed aniline by G. Bengtsson; C. Carlsson (pp. 234-241).

The influence of sorption on the mineralisation of 50 µg aniline l^–1 was examined in an aquifer material under batch conditions. The study was designed to distinguish the rates and extent of biodegradation of the sorbed and the dissolved trace organic and the contribution of sorbed and suspended bacteria to the degradation. Four different mathematical models were developed with different assumptions about the partitioning of aniline degradation and bacterial activity between the solid and the aqueous phases. The models were developed by combining an expression for logistic growth of the degrading population with Michaelis–Menten kinetics for the transformation of aniline. It was tested by a series of laboratory experiments conducted with ¹⁴C-labelled aniline, aseptically treated aquifer sand and filter-sterilised groundwater in different proportions and bacteria isolated from pristine groundwater. Model evaluation of the experimental data suggested that the fate of aniline was mainly controlled by suspended bacteria degrading both the dissolved and sorbed fractions. The degradation was slow, with a first-order degradation rate equal to 10^–6 h^–1.

On site bioremediation of hydrocarbon-contaminated Arctic tundra soils in inoculated biopiles by W. Mohn; C. Radziminski; M.-C. Fortin; K. Reimer (pp. 242-247).

There is a need to develop technology to allow the remediation of soil in polar regions that have been contaminated by hydrocarbon fuel spills. Bioremediation is potentially useful for this purpose, but has not been well demonstrated in polar regions. We investigated biopiles for on-site bioremediation of soil contaminated with Arctic diesel fuel in two independent small-scale field experiments at different sites on the Arctic tundra. The results were highly consistent with one another. In biopiles at both sites, extensive hydrocarbon removal occurred after one summer. After 1 year in treatments with optimal conditions, total petroleum hydrocarbons were reduced from 196 to below 10 mg per kg of soil at one site, and from 2,109 to 195 mg per kg of soil at the other site. Addition of ammonium chloride and sodium phosphate greatly stimulated hydrocarbon removal and indicates that biodegradation was the primary mechanism by which this was achieved. Inoculation with cold-adapted, mixed microbial cultures further stimulated hydrocarbon removal during the summer immediately following inoculation. At one site, soil temperature was monitored during the summer season, and a clear plastic cover increased biopile soil temperature, measured as degree-day accumulation, by 30–49%. Our results show that on-site bioremediation of fuel-contaminated soil at Arctic tundra sites is feasible.

Toward the bioremediation of dioxin-polluted soil: structural and functional analyses of in situ microbial populations by quinone profiling and culture-dependent methods by A. Hiraishi; H. Miyakoda; B.-R. Lim; H.-Y. Hu; K. Fujie; J. Suzuki (pp. 248-256).

In order to obtain basic information toward the bioremediation of dioxin-polluted soil, microbial communities in farmland soils polluted with high concentrations of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were studied by quinone profiling as well as conventional microbiological methods. The concentration of PCDD/Fs in the polluted soils ranged from 36 to 4,980 pg toxicity equivalent quality (TEQ) g^–1 dry weight of soil. There was an inverse relationship between the levels of PCDD/Fs and microbial biomass as measured by direct cell counting and quinone profiling. The most abundant quinone type detected was either MK-6 or Q-10. In addition, MK-8, MK-8(H₂), and MK-9(H₈) were detected in significant amounts. Numerical analysis of quinone profiles showed that the heavily polluted soils (≥1,430 pg TEQ g^–1) contained different community structures from lightly polluted soils (≤56 pg TEQ g^–1). Cultivation of the microbial populations in the heavily polluted soils with dibenzofuran or 2-chlorodibenzofuran resulted in enrichment of Q-10-containing bacteria. When the heavily polluted soil was incubated in static bottles with autoclaved compost as an organic nutrient additive, the concentrations of PCDD/Fs in the soil were decreased by 22% after 3 months of incubation. These results indicate that dioxin pollution exerted a significant effect on microbial populations in soil in terms of quantity, quality, and activity. The in situ microbial populations in the dioxin-polluted soil were suggested to have a potential for the transformation of PCDD/Fs and oxidative degradation of the lower chlorinated ones thus produced.

Chromate reduction and 16S rRNA identification of bacteria isolated from a Cr(VI)-contaminated site by P. Pattanapipitpaisal; N. Brown; L. Macaskie (pp. 257-261).

A Gram-positive, hexavalent chromium [chromate: Cr(VI)]-tolerant bacterium, isolated from tannery waste from Pakistan, was identified as a Microbacterium sp. by 16S rRNA gene sequence homology. The strain (designated as MP30) reduced toxic Cr(VI) only under anaerobic conditions at the expense of acetate as the electron donor. The bacterium was able to grow aerobically in L-broth supplemented with 15 mM CrO₄ ^2– but then did not reduce Cr(VI). At a concentration of 2.4×10⁹ cells/ml, 100 µM sodium chromate was reduced within 30 h; however, the maximum specific reduction rate was obtained at lower initial cell concentrations.

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Applied Microbiology and Biotechnology (v.57, #1-2)