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Applied Microbiology and Biotechnology (v.51, #1)
Bacterial catabolic transposons by H.-M. Tan (pp. 1-12).
The introduction of foreign organic hydrocarbons into the environment in recent years, as in the widespread use of antibiotics, has resulted in the evolution of novel adaptive mechanisms by bacteria for the biodegradation of the organic pollutants. Plasmids have been implicated in the catabolism of many of these complex xenobiotics. The catabolic genes are prone to undergo genetic rearrangement and this is due to their presence on transposons or their association with transposable elements. Most of the catabolic transposons have structural features of the class I (composite) elements. These include transposons for chlorobenzoate (Tn5271), chlorobenzene (Tn5280), the newly discovered benzene catabolic transposon (Tn5542), and transposons encoding halogenated alkanoates and nylon-oligomer-degradative genes. Transposons for the catabolism of toluene (Tn4651, Tn4653, Tn4656) and naphthalene (Tn4655) belong to class II (Tn3 family) elements. Many catabolic genes have been associated with insertion sequences, which suggests that these gene clusters could be rapidly disseminated among the bacterial populations. This greatly expands the substrate range of the microorganisms in the environment and aids the evolution of new and novel degradative pathways. This enhanced metabolic versatility can be exploited for and is believed to play a major part in the bioremediation of polluted environments.
Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation by J. Choi; S. Y. Lee (pp. 13-21).
Polyhydroxyalkanoates (PHA) have been attracting considerable attention as biodegradable substitutes for conventional polymers. To reduce their production cost, a great deal of effort has been devoted to developing better bacterial strains and more efficient fermentation/recovery processes. In this paper, several factors affecting the production cost of PHA, such as PHA productivity, content and yield, the cost of the carbon substrate, and the recovery method were reviewed. A sensitivity analysis was also carried out with respect to these factors and with a view to scale-up. Several production processes were designed on the basis of the reported fermentation and recovery results, and were economically evaluated. PHA productivity only affects equipment-related costs, but PHA content has multiple effects on the process economics. Development of an economical and efficient recovery method is also important to the overall economics of PHA production.
Rhamnose lipids – biosynthesis, microbial production and application potential by S. Lang; D. Wullbrandt (pp. 22-32).
Biosurfactants containing rhamnose and β-hydroxydecanoic acid and called rhamnolipids are reviewed with respect to microbial producers, their physiological role, biosynthesis and genetics, and especially their microbial overproduction, physicochemical properties and potential applications. With Pseudomonas species, more than 100 g l−1 rhamnolipids were produced from 160 g l−1 soybean oil at a volumetric productivity of 0.4 g l−1 h−1. The individual rhamnolipids are able to lower the surface tension of water from 72 mN m−1 to 25–30 mN m−1 at concentrations of 10–200 mg l−1. After initial testing, rhamnolipids seem to have potential applications in combating marine oil pollution, removing oil from sand and in combating zoosporic phytopathogens. Rhamnolipids are also a source of l-rhamnose, which is already used for the industrial production of high-quality flavor components.
Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources by S. Spoeckner; V. Wray; M. Nimtz; S. Lang (pp. 33-39).
When grown on vegetable oils and their derivatives, the smut fungus Ustilago maydis (DSM 4500 and ATCC 14826) produces several glycolipids under nitrogen-limiting conditions. With 45 g l−1 sunflower oil fatty acids (technical grade) a yield of 30 g l−1 glycolipid was achieved. The resulting mixture contained predominantly mannosylerythritol lipids together with smaller amounts of cellobiose lipids. The production of the more polar cellobiose lipids was enhanced when glucose was used as carbon source. The molecular structure of the main components of the glycolipid mixture were elucidated by a combination of NMR spectroscopic and mass-spectrometric techniques.
Production of sophorolipids from whey: development of a two-stage process with Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214 using deproteinized whey concentrates as substrates by H.-J. Daniel; R. T. Otto; M. Binder; M. Reuss; C. Syldatk (pp. 40-45).
In order to produce sophorolipids from whey, thereby lowering the lactose content and biological oxygen demand, a two-step batch cultivation process was developed including medium sterilization by filtration. In the first step, whey was sterilized by a combination of crossflow and sterile filtration. Because the sophorolipid-producing yeast Candida bombicola ATCC 22214 was not able to use lactose as a carbon source directly, the oleaginous yeast Cryptococcus curvatus ATCC 20509 was grown on deproteinized whey concentrates (DWC). With 1: 1 diluted DWC-20, lactose was consumed as the carbon source and biomass (24 g/l dry weight content) as well as single-cell oil (SCO, 10 g/l) were produced. The cultivation broth was disrupted with a glass bead mill and it served as medium for growth (29 g cell dry mass/l) and sophorolipid production (12 g/l) of the yeast C. bombicola.
Gratuitous dechlorination of chloroethanes by methanogenic granular sludge by M. H. A. van Eekert; A. J. M. Stams; J. A. Field; G. Schraa (pp. 46-52).
The dechlorinating activity of a methanogenic granular sludge from a methanol-fed upflow anaerobic sludge blanket reactor was investigated with chlorinated ethanes. This unadapted methanogenic consortium degraded all chloroethanes tested. The product formation rates decreased with the number of chlorine substituents. The more highly chlorinated ethanes were also converted, although at a lower rate, in the presence of autoclaved (dead) sludge, indicating the involvement of reduced heat-stable cofactors like vitamin B12 and F430. Direct chemical dechlorination of hexa-, penta- and tetrachloroethanes was also observed in medium without sludge, although at a much lower rate. The results show the importance of cometabolic and abiotic (chemical) conversions for the transformation of chlorinated ethanes by the methanogenic consortium. The types of reaction and the products formed were correlated with the Gibbs free-energy change (ΔG 0′). Reductive hydrogenolysis and dichloroelimination were important dechlorinating mechanisms. Generally, these reactions have a higher ΔG 0′ value than dehydrochlorination reactions, which occurred less frequently during the transformation of chloroethanes by the methanogenic granular sludge.
Production of (R )-3-pentyn-2-ol through stereospecific hydrolysis of racemic 3-pentyn-2-ol esters with microbial enzymes by J. Ogawa; S.-X. Xie; S. Shimizu (pp. 53-57).
Microorganisms and commercial enzymes were screened for their ability to produce (R)-3-pentyn-2-ol from racemic 3-pentyn-2-ol esters through stereospecific hydrolysis. Among the esters formed with acetic acid, propionic acid, hexanoic acid and benzoic acid, the acetate was most effectively hydrolyzed by microbial cells and commercial lipases with high stereospecificity. Rhodococcus rubropertinctus AKU NOC082 was a good catalyst for (R)-3-pentyn-2-ol production through the hydrolytic resolution of racemic 3-pentyn-2-yl acetate. With 15%, 25% and 50% (v/v) racemic 3-pentyn-2-yl acetate as the substrate, 42.6%, 40.8% and 40.0% was hydrolyzed in 5 h, 10 h and 98 h respectively, under the optimized conditions (pH 7.0, 30 °C, 7.5% wet cell concentration), the (R) enantiomer of 3-pentyn-2-ol being formed with an optical purity of 97.8%, 98.0% and 94.2% respectively.
Characterization of a glucose 3-dehydrogenase from the cultivated mushroom (Agaricus bisporus ) by S. C. Morrison; D. A. Wood; P. M. Wood (pp. 58-64).
An extracellular enzyme with glucose dehydrogenase activity was purified from liquid cultures of the basidiomycete Agaricus bisporus after growth with d-cellobiose or d-glucose as carbon source. The molecular mass was measured as 57 kDa by gel filtration and 55 kDa by sodiumdodecyl sulphate/polyacrylamide gel electrophoresis, while the isoelectric point was at pH 3.6. By analysis of 1H-NMR spectra in D2O, the product of d-glucose oxidation was identified as 3-ketoglucose. The substrates oxidized included d-cellobiose, l-arabinose, d-xylose and sucrose, but the specificity parameter (k cat/K m) was highest for d-glucose. Two electron acceptors were identified, namely 2,6-dichloroindophenol and p-benzoquinone, but reduction of dioxygen, ferricyanide or cytochrome c was not detectable. The selective C-3 oxidation of d-glucose is well-characterized for Agrobacterium and Flavobacterium, but this is the first report for a fungus.
Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface by T. Murai; M. Ueda; Y. Shibasaki; N. Kamasawa; M. Osumi; T. Imanaka; A. Tanaka (pp. 65-70).
The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase.
The expression signals of the Lactobacillus brevis slpA gene direct efficient heterologous protein production in lactic acid bacteria by M. Kahala; A. Palva (pp. 71-78).
A cassette based on the expression signals of the Lactobacillus brevis surface (S)-layer protein gene (slpA) was constructed. The low-copy-number vector pKTH2095, derived from pGK12, was used as the cloning vector. The efficiency of slpA promoters in intracellular protein production was studied using three reporter genes, β-glucuronidase (gusA), luciferase (luc) and aminopeptidase N (pepN) in three different lactic acid bacteria hosts: Lactococcus lactis, Lactobacillus plantarum and Lactobacillus gasseri. The S-layer promoters were recognized in each strain and especially L. lactis and Lb. plantarum exhibited high levels of transcripts. The production kinetics of reporter proteins was studied as a function of growth. The GusA, Luc and PepN activities varied considerably among the lactic acid bacterial strains studied. The highest levels of β-glucuronidase and luciferase activity were obtained in L. lactis. The level of GusA obtained in L. lactis corresponded to over 15% of the total cellular proteins. The highest level of aminopeptidase N activity was achieved in Lb. plantarum where PepN corresponded up to 28% of the total cellular proteins at the late exponential phase of growth. This level of PepN activity is 30-fold higher than that in Lb. helveticus, which is the species from which the pepN gene originates.
Efficient expression of mosquito-larvicidal proteins in a gram-negative bacterium capable of recolonization in the guts of Anopheles dirus larva by P. Khampang; W. Chungjatupornchai; P. Luxananil; S. Panyim (pp. 79-84).
The gram-negative bacterium, An11/2 G1, isolated from the guts of Anopheles dirus mosquito larvae, was identified as Enterobacter amnigenus. The E. amnigenus was able to recolonize in the gut of An. dirus larva but not in those of Aedes aegypti and Culex quinquefasciatus larvae. It was able to float in water for a longer period than Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus. These are desirable characteristics for a delivery vehicle of mosquito-larvicidal toxins for the control of mosquito larvae, and E. amnigenus was therefore used as a host to express the cryIVB gene of B. thuringiensis subsp. israelensis and the binary toxin genes of B. sphaericus. The recombinant E. amnigenus produced a high level of CryIVB protein, which was toxic to larvae of Ae. aegypti and An. dirus. Another E. amnigenus producing the 51-kDa protein of B. sphaericus was toxic to larvae of An. dirus and Cx. quinquefasciatus. The recombinant plasmids were stable in E. amnigenus without the presence of selective pressure for at least 23 generations. The recombinant E. amnigenus should represent a desirable biological agent for controlling mosquito larvae.
A novel mutation of the Bacillus subtilis hut operon that relieves both catabolite repression and amino acid repression by S. Eda; T. Hoshino; M. Oda (pp. 85-90).
A mutation, designated hutCR11, which resulted in high expression of the hut operon and release of the catabolite repression and amino-acid repression of hut expression, was isolated and determined to be a T-to-G transversion at position +30 (+1 indicates the transcription-initiation site). In the hutCR11 mutant, levels of hutP mRNA were 5-fold higher than those in wild-type cells under conditions of non-induction and induction and 11-fold higher under conditions of catabolite repression and amino-acid repression. Mutation analysis showed that two types of base change (T → A and T → C) at position +30 did not cause high expression of the hut operon, indicating that this was specifically caused by the single base substitution (T → G) at position +30. The base substitution of A for T at position +30 also led to partial relief of both catabolite repression and amino-acid repression. These results indicate that the nucleotide sequence at +30 is important for regulation of both catabolite repression and amino-acid repression of the hut operon.
Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates by D. Corroler; N. Desmasures; M. Gueguen (pp. 91-99).
A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L.lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency.
Mannitol-enhanced survival of Lactococcus lactis subjected to drying by B. J. O. Efiuvwevwere; L. G. M. Gorris; E. J. Smid; E. P. W. Kets (pp. 100-104).
Survival of Lactococcus lactis subjected to different drying conditions was investigated. Mannitol most remarkably enhanced the survival of dried cells to a level almost equalling that of viable cells [log10 (cfu ml−1) = 9.42] as was found prior to the drying process (log10 = 9.6). In the absence of mannitol, a survival was reduced by a factor of 104. Drying of cells at 20 °C led to higher survival rates than drying at 30 °C. Mannitol enhanced the survival rate at both temperatures, and at both 20 °C and 30 °C the highest reduction in survival occurred when cells were dried at a water activity of 0.76. In the presence of mannitol, differences in survival after drying at different water activities were less pronounced. Rehydration of cells dried in the presence of mannitol resulted in an extended lag phase of 4 h compared to fresh cells. No growth or acidification of the culture medium was observed for 12 h in the case of rehydrated cells dried in the absence of mannitol. It was hypothesized that a radical scavenging activity of mannitol could partly explain these observations.
Bioavailability of hydrocarbons during microbial remediation of a sandy soil by C. Löser; H. Seidel; P. Hoffmann; A. Zehnsdorf (pp. 105-111).
The microbial degradation of hydrocarbons was studied in an artificially contaminated sandy soil, using a pilot-scale percolator system. After a short lag period, an intensive degradation occurred, which diminished in time and completely stopped in the end, despite large residual contaminations (residues of 56% diesel fuel, 20% n-hexadecane and 3.5% phenanthrene at the initial loadings of each 3000 mg/kg). The remaining pollutant content was influenced by the kind of hydrocarbon but was nearly independent of its initial loading. According to a model-aided analysis of the carbon dioxide production during remediation, the observed stagnation of degradation was caused by a limited bioavailability of the pollutants. The degradation in the soil-free aqueous phase was more extensive than in the soil, which suggests that the limited bioavailability in the soil can be attributed mainly to matrix-dependent rather than substrate-dependent influences. Generally, fine particles and organic matter are mainly responsible for the adsorption of pollutants to the soil matrix. Our sandy soil also bound hydrocarbons adsorptively although it contained neither silty material nor significant amounts of organic matter. As shown by Brunauer Emmett Teller (BET) analysis, the soil particles were covered by micropores, which enlarged the soil surface by a factor of 120 in comparison with the macroscopic surface area. The microporosity is the reason for the hydrocarbons being more strongly adsorbed to the sandy soil than expected.
Disposable sensor for measuring the biochemical oxygen demand for nitrification and inhibition of nitrification in wastewater by A. König; T. T. Bachmann; J. W. Metzger; R. D. Schmid (pp. 112-117).
A disposable-type microbial sensor was developed for the determination of both the biochemical oxygen demand for nitrification (N-BOD) and inhibiting effects on nitrifying bacteria. The sensor was based on the respiratory activity of nitrifying bacteria immobilized on a miniature oxygen electrode. Typical response times for measuring N-BOD of ammonium standard solutions as well as of wastewater samples were in the range of 6–12 min. A dynamic evaluation of the signals after a measuring time of 120 s also resulted in good reproducibility and sensitivity. A daily profile of a municipal sewage plant was recorded, comparing the biosensor data with two standard methods. For the measurement of nitrification-inhibiting effects a 120-s dynamic signal evaluation was preferred to a steady-state method because of the long recovery times resulting from extended exposure to inhibitors. However, steady-state measurement techniques allowed allylthiourea detection with a ten times higher sensitivity. Because of the advantages of this miniaturized electrode, e.g. short response time, simple measuring procedure and low costs of production, this sensor system is considered to be suitable for commercial application in environmental analysis.