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Applied Microbiology and Biotechnology (v.66, #1)
Applications of biopolymers and other biotechnological products in building materials by Johann Plank (pp. 1-9).
Bio admixtures are functional molecules used in building products to optimize material properties. They include natural or modified biopolymers, biotechnological and biodegradable products. Concrete and dry-mix mortars (e.g. wall plasters or tile adhesives) represent two major applications for bio admixtures. Examples of bio products used in concrete are lignosulfonate, sodium gluconate, pine root extract, protein hydrolysates and Welan gum; and in dry-mix mortar methyl hydroxypropyl cellulose, hydroxypropyl starch, guar gum, tartaric acid, casein, succinoglycan and Xanthan gum. In a number of applications, bio admixtures compete well with synthetic admixtures. Sometimes, they are indispensable in the formulation of certain building products. Their market share is expected to increase because of technological advances, particularly in the field of microbial biopolymers, and because of the growing trend to use naturally based or biodegradable products in building materials.
Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass by H. B. Klinke; A. B. Thomsen; B. K. Ahring (pp. 10-26).
An overview of the different inhibitors formed by pre-treatment of lignocellulosic materials and their inhibition of ethanol production in yeast and bacteria is given. Different high temperature physical pre-treatment methods are available to render the carbohydrates in lignocellulose accessible for ethanol fermentation. The resulting hydrolyzsates contain substances inhibitory to fermentation—depending on both the raw material (biomass) and the pre-treatment applied. An overview of the inhibitory effect on ethanol production by yeast and bacteria is presented. Apart from furans formed by sugar degradation, phenol monomers from lignin degradation are important co-factors in hydrolysate inhibition, and inhibitory effects of these aromatic compounds on different ethanol producing microorganisms is reviewed. The furans and phenols generally inhibited growth and ethanol production rate (QEtOH) but not the ethanol yields (YEtOH) in Saccharomyces cerevisiae. Within the same phenol functional group (aldehyde, ketone, and acid) the inhibition of volumetric ethanol productivity was found to depend on the amount of methoxyl substituents and hence hydrophobicity (log P). Many pentose-utilizing strains Escherichia coli, Pichia stipititis, and Zymomonas mobilis produce ethanol in concentrated hemicellulose liquors but detoxification by overliming is needed. Thermoanaerobacter mathranii A3M3 can grow on pentoses and produce ethanol in hydrolysate without any need for detoxification.
Enzymatic enantioselective transcyanation of silicon-containing aliphatic ketone with (S)-hydroxynitrile lyase from Manihot esculenta by Ruo Xu; Min-Hua Zong; Yu-Ying Liu; Jun He; Yuan-Yuan Zhang; Wen-Yong Lou (pp. 27-33).
(S)-Hydroxynitrile lyase from Manihot esculenta (MeHNL) was shown for the first time to be able to catalyze the enantioselective transcyanation of acetyltrimethylsilane (ATMS) with acetone cyanohydrin to form (S)-2-trimethylsilyl-2-hydroxyl-propionitrile in an aqueous/organic biphasic system. To better understand the reaction, various influential variables were examined. The most suitable organic phase, optimal buffer pH, aqueous phase content, shaking rate, temperature, concentration of ATMS, acetone cyanohydrin and crude enzyme were diisopropyl ether (DIPE), 5.4, 13% (v/v), 190 rpm, 40°C, 10 mM, 20 mM, and 35 U/ml, respectively, under which the initial reaction rate, substrate conversion and product enantiomeric excess (e.e.) were 19.5 mM/h, 99.0% and 93.5%, respectively. A comparative study demonstrated that silicon atoms in the substrate had a great effect on the reaction, and that ATMS was a much better substrate for MeHNL than its carbon analogue 3,3-dimethyl-2-butanone (DMBO) with respect to the initial reaction rate, substrate conversion and product e.e. MeHNL has greater affinity towards ATMS than its carbon analogue as indicated by the much lower Km. The activation energy of MeHNL-catalyzed transcyanation of ATMS was also markedly lower than that of DMBO. The silicon effect on the reaction was rationalized on the basis of the special characteristics of silicon atoms and the catalytic mechanism of MeHNL.
An enzymatic route to produce pyruvate from lactate by C. Q. Ma; P. Xu; J. H. Qiu; Z. J. Zhang; K. W. Wang; M. Wang; Y. N. Zhang (pp. 34-39).
A bacterial strain of Acinetobacter sp., which was capable of enzymatic production of pyruvate from lactate, was cultured in a 5-l reactor with a basal salt medium. After 14 h of fed-batch fermentation, 9.56 g l−1 cell concentration in the broth was obtained with 20 g l−1 (178 mM) sodium lactate and 4 g l−1 NH4Cl in the medium; and the biotransformation ability was 2.51 units ml−1. The cells were harvested from one reactor and then used for pyruvate production from lactate in the same reactor. l-lactate at a concentration about 527 mM was almost stoichiometrically converted to pyruvate in 28 h. After a total 42 h of cell culture and biotransformation, the transformative yield was about 0.72 g g−1 pyruvate from lactate and the rate of pyruvate production was calculated as 1.33 g l−1 h−1 during the process. The results suggested this simple enzymatic production of pyruvate from lactate should be a promising process and may bring a yield higher than that by microbial fermentation. By this process, the recovery of pyruvate from such a simple reaction liquid is relatively easy and inexpensive to perform.
Biodegradation of hydrocarbon cuts used for diesel oil formulation by Sophie Penet; Rémy Marchal; Abdelghani Sghir; Frédéric Monot (pp. 40-47).
The biodegradability of various types of diesel oil (DO), such as straight-run DO, light-cycle DO, hydrocracking DO, Fischer–Tropsch DO and commercial DO, was investigated in biodegradation tests performed in closed-batch systems using two microflorae. The first microflora was an activated sludge from an urban wastewater treatment plant as commonly used in biodegradability tests of commercial products and the second was a microflora from a hydrocarbon-polluted soil with possible specific capacities for hydrocarbon degradation. Kinetics of CO2 production and extent of DO biodegradation were obtained by chromatographic procedures. Under optimised conditions, the polluted-soil microflora was found to extensively degrade all the DO types tested, the degradation efficiencies being higher than 88%. For all the DOs tested, the biodegradation capacities of the soil microflora were significantly higher than those of the activated sludge. Using both microflora, the extent of biodegradation was highly dependent upon the type of DO used, especially its hydrocarbon composition. Linear alkanes were completely degraded in each test, whereas identifiable branched alkanes such as farnesane, pristane or phytane were degraded to variable extents. Among the aromatics, substituted mono-aromatics were also variably biodegraded.
Enhanced production of lactococcin 972 in chemostat cultures by Alma Hernández de Rojas; Beatriz Martínez; Juan E. Suárez; Ana Rodríguez (pp. 48-52).
Lactococcus lactis subsp. lactis IPLA972 is a wild lactococcal strain suitable as a single starter in the manufacture of dairy products. This strain synthesizes lactococcin 972 (Lcn972), a unique bacteriocin that blocks septum formation. In this work, we report on the conditions to optimize biomass and Lcn972 production. In batch cultures, pH 6.8 was found to be optimum for bacteriocin synthesis and both glucose and lactose supported Lcn972 production. The inhibitory activity improved up to eight-fold with increasing carbohydrate concentration. In chemostat cultures, steady states were achieved even at dilution rates higher than μ max, due to the strong wall growth. Lcn972 behaved as a true primary metabolite, as it was maximally produced when the cells were actively growing. Bacteriocin yields were improved up to ten-fold in chemostat cultures compared with those achieved in batch.
Purification and characterization of NADPH-dependent aldo–keto reductase specific for β-keto esters from Penicillium citrinum, and production of methyl (S)-4-bromo-3-hydroxybutyrate by N. Itoh; H. Asako; K. Banno; Y. Makino; M. Shinohara; T. Dairi; R. Wakita; M. Shimizu (pp. 53-62).
A novel β-keto ester reductase (KER) was purified to homogeneity from recombinant Escherichia coli (pTrcKER) cells, which efficiently expressed the ker gene cloned from Penicillium citrinum IFO4631. The enzyme was monomeric and had a molecular mass of 37 kDa. It catalyzed the reduction of some β-keto esters, especially alkyl 4-halo-3-oxobutyrates. However, it did not catalyze the reverse reaction, the dehydrogenation of alkyl 4-halo-3-hydroxybutyrates and other alcohols. The enzyme required NADPH as a cofactor and showed no activity with NADH. Therefore, it was defined as a NADPH-dependent aldo–keto reductase (AKR3E1), belonging to the AKR superfamily. The enzyme stereospecifically produced methyl (S)-4-bromo-3-hydroxybutyrate from its keto derivative with high stereospecificity (97.9% enantiomer excess). E. coli cells expressing KER and glucose dehydrogenase in the water/butyl acetate two-phase system achieved a high productivity of (S)-4-bromo-3-hydroxybutyrate (277 mM, 54 mg/ml) in the organic solvent layer.
Keywords: Aldo–keto reductase (NADPH)Penicillium citrinumBiocatalyst; β-Keto ester; Methyl (S)-4-bromo-3-hydroxybutyrate
Characterization and molecular cloning of a novel endoglucanase from Trichoderma sp. C-4 by Ok-Ju Sul; Ji-Hyun Kim; Sun-Ju Park; Young-Jun Son; Bo-Ryung Park; Dae Kyun Chung; Choon-Soo Jeong; In-Seob Han (pp. 63-70).
A fungal strain, C-4, was isolated from etiolated leaves. Based on taxonomic studies, the fungus C-4 can be classified as a strain of Trichoderma species. When strain C-4 was cultured in Mandels’ medium at 28°C for 6 days, the enzyme activities detected in the broth corresponded to 8.2 U/ml (28.1 U/mg) carboxymethylcellulase activity. An endoglucanase (EG; F-I-II) was purified from the culture filtrate of the strain through a four-step procedure—chromatography on Sephacryl S-200, DEAE-Sephadex A-50, Con A-Sepharose, and Chromatofocusing on Mono-P (HPLC). The molecular weight of this EG, which was called C4endoII, was determined to be about 51 kDa. The optimum temperature and pH of C4endoII were 50°C and 5.0, respectively. Incubation at 50°C for 24 h did not destroy the cellulose degradation activity. Amino acid sequence analysis revealed the N-terminal sequence of an internal peptide of C4endoII to be Phe-Ala-Gly-Ile-Asn-Ile-Ala-Gly-Phe-Asp-Phe, which is homologous to EGII from Trichoderma reesei. A C4endoII cDNA (C4endoII) was cloned from a cDNA library constructed using the mRNA of the strain cultivated in a cellulase-induction medium. The deduced protein sequence of C4endoII was 417 amino acids long and had a putative signal sequence of 21 amino acids with a predicted cleavage site after Ala-21. A single potential N-glycosylation site was present in the amino acid sequence.
Asparaginase inhibition of adhesion of type 1-fimbriated and P-fimbriated Escherichia coli to epithelial cell receptors by M. R. Wehner; S. R. Koch; L. Kindinger; N. Nelson; M. M. Cowan (pp. 71-73).
The 3D structures of Fim H and PapG proteins complexed with the host carbohydrate receptor demonstrate that both utilize binding-pocket asparagines for contact or stabilization with the carbohydrate. Pretreatment of whole bacteria with asparaginase resulted in decreased fimbriae-mediated attachment to urinary epithelial cells. Enzyme treatment of bacteria pre-adhered to epithelial cells removed more uropathogenic E. coli than the indigenous flora attached to them.
Mutation study of conserved amino acid residues of Spirulina Δ6-acyl-lipid desaturase showing involvement of histidine 313 in the regioselectivity of the enzyme by Apiradee Hongsthong; Sanjukta Subudhi; Matura Sirijuntarat; Supapon Cheevadhanarak (pp. 74-84).
In the cyanobacterium Spirulina platensis, the desaturation process is carried out by three desaturases: the Δ9, Δ12 and Δ6 desaturases, encoded by desC, desA and desD, respectively. The Δ6 desaturase is responsible for the catalysis of linoleic acid, yielding γ-linolenic acid (18:3Δ9,12,6), the end-product of the process. In this study, the desD gene was expressed in Escherichia coli using a pTrcHisA expression system. In order to identify the amino acid residues involved in the enzymatic activity, a sequence comparison was performed using various organisms. The alignment revealed three conserved histidine clusters, a number of conserved residues among all listed organisms and a few conserved residues among cyanobacterial species possibly involved in the desaturation activity. A series of site-directed mutations were generated in the desD gene to evaluate the role of these residues vis-à-vis the enzyme function. This approach revealed that: (1) H313 is involved in the regioselectivity of the enzyme, (2) the three histidine clusters together with H313, H315, D138 and E140 are required for enzymatic activity, most likely as providers of the catalytic Fe center and (3) W294 is also essential for the activity of Δ6 desaturase, possibly by forming part of the substrate-binding pocket.
Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae by Eduardo Rodriguez; Shannon Ward; Hong Fu; W. Peter Revill; Robert McDaniel; Leonard Katz (pp. 85-91).
Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors—malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA—used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG–K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1 g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors.
Degradation pathways of cyclic alkanes in Rhodococcus sp. NDKK48 by D. Koma; Y. Sakashita; K. Kubota; Y. Fujii; F. Hasumi; S. Y. Chung; M. Kubo (pp. 92-99).
The degradation pathways for cyclic alkanes (c-alkanes) in Rhodococcus sp. NDKK48 were investigated. Strain NDKK48 used dodecylcyclohexane as a sole carbon and energy source, and five metabolites in the dodecylcyclohexane degradation pathway were detected by gas-chromatography/mass spectra. The metabolites were identified as cyclohexanecarboxylic acid, cyclohexylacetic acid, 1-cyclohexene-1-acetic acid, 4-dodecylcyclohexanol, and 4-dodecylcyclohexanone. The strain degrades dodecylcyclohexane via a ring oxidation pathway and an alkyl side chain oxidation pathway. Cyclohexanecarboxylic acid was further oxidized to muconic acid via 1-cyclohexene-1-carboxylic acid and benzoic acid, and the muconic acid was finally used by strain NDKK48 for growth. Methylcyclohexane and cyclohexane were co-oxidized with hexadecane by strain NDKK48. Methylcyclohexane was degraded via a ring oxidation pathway, and the degradation pathway contained part of the Baeyer-Villiger oxidation for ring cleavage. Cyclohexane was also degraded by the same pathway as methylcyclohexane. Thus, strain NDKK48 has two pathways for the complete degradation of c-alkanes.
Microbial composition of biofilms in a brewery investigated by fatty acid analysis, fluorescence in situ hybridisation and isolation techniques by Markus Timke; Dorothee Wolking; Ngoc Quynh Wang-Lieu; Karlheinz Altendorf; André Lipski (pp. 100-107).
Biofilms associated with brewery plants can harbour spoiling microorganisms that potentially damage the final product. Most beer-spoiling microorganisms are thought to depend on numerous interactions with the accompanying microbiota. However, there is no information on the microbial community structure of biofilms from bottling plants. The conveyors that transport the bottles to and from the plant are known as potential sources of microbial contamination of beer. Consequently, the material buildup from two conveyors was analysed using a cultivation/isolation approach, and the culture-independent techniques of whole cell fatty acid analysis and fluorescence in situ hybridisation (FISH). Heterogeneous communities were present at both conveyors. Although characteristic fatty acids for Eukarya were present, FISH-signals for Eukarya were extremely low. The Proteobacteria, in particular the Gammaproteobacteria, were abundant at both sample sites. Bacterial isolates were obtained for every dominating group detected by FISH: the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, the Xanthomonadaceae, the Actinobacteria, the Bacteroidetes and the Firmicutes.
Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells by Shingo Izawa; Machiko Sato; Kumio Yokoigawa; Yoshiharu Inoue (pp. 108-114).
Glycerol is well known as a cryoprotectant similar to trehalose. However, there is little information about the effects of intracellular glycerol on the freeze-thaw stress tolerance of yeast. Through analysis of a quadruple-knockout mutant of glycerol dehydrogenase genes (ara1Δ gcy1Δ gre3Δ ypr1Δ) in Saccharomyces cerevisiae, we revealed that the decrease in glycerol dehydrogenase activity led to increased levels of intracellular glycerol. We also found that this mutant showed higher tolerance to freeze stress than wild type strain W303-1A. Furthermore, we demonstrated that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability in dough even after frozen storage for 7 days. These results suggest the possibility of using intracellular-glycerol-enriched cells to develop better frozen dough.
Analysis of the bacterial community inhabiting an aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste by P. Juteau; D. Tremblay; R. Villemur; J.-G. Bisaillon; R. Beaudet (pp. 115-122).
The microflora of a self-heating aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste was investigated by a combination of culture and culture-independent techniques. The temperature increased quickly in the first hours of the treatment cycles and values up to 72°C were reached. Denaturing gradient gel electrophoresis of the PCR-amplified V3 region of 16S rDNA (PCR-DGGE) revealed important changes in the bacterial community during 3-day cycles. A clone library was constructed with the near-full-length 16S rDNA amplified from a mixed-liquor sample taken at 60°C. Among the 78 non-chimeric clones analysed, 20 species (here defined as clones showing more than 97% sequence homology) were found. In contrast to other culture-independent bacterial analyses of aerobic thermophilic wastewater treatments, species belonging to the Bacilli class were dominant (64%) with Bacillus thermocloacae being the most abundant species (38%). The other Bacilli could not be assigned to a known species. Schineria larvae was the second most abundant species (14%) in the clone library. Four species were also found among the 19 strains isolated, cultivated and identified from samples taken at 40°C and 60°C. Ten isolates showed high 16S rDNA sequence homology with the dominant bacterium of a composting process that had not been previously isolated.