Eight fungal species known to produce wood pigmentation were tested for reaction to various moisture contents in two hardwood species. Fungal pigmentation by Trametes versicolor and Xylaria polymorpha was stimulated at low water concentrations in both Acer saccharum (sugar maple) and Fagus grandifolia (American beech), while Inonotus hispidus and Polyporus squamosus were stimulated above 22-28% and 34-38% moisture content in beech and in sugar maple respectively. Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26 - 41% and in sugar maple at 59 - 96% moisture content. The pink staining Scytalidium cuboideum pigmented both wood species at above 35% moisture content. This research indicates that controlling the moisture content values of wood substrates can stimulate the intensity of pigmentation of specific fungi when spalting wood for decorative and commercial purpose.
Fungal melanin; Pigment; Moisture content; Spalting
Spent Sulfite Liquor (SSL) from wood pulping facilities is a sugar rich effluent that can be used as feedstock for ethanol production. However, depending on the pulping process conditions, the release of monosaccharides also generates a range of compounds that negatively affect microbial fermentation. In the present study, we investigated whether endogenous yeasts in SSL-based ethanol plant could represent a source of Saccharomyces cerevisiae strains with a naturally acquired tolerance towards this inhibitory environment. Two isolation processes were performed, before and after the re-inoculation of the plant with a commercial baker’s yeast strain. The isolates were clustered by DNA fingerprinting and a recurrent Saccharomyces cerevisiae strain, different from the inoculated commercial baker’s yeast strain, was isolated. The strain, named TMB3720, flocculated heavily and presented high furaldehyde reductase activity. During fermentation of undiluted SSL, TMB3720 displayed a 4-fold higher ethanol production rate and 1.8-fold higher ethanol yield as compared to the commercial baker’s yeast. Another non-Saccharomyces cerevisiae species, identified as the pentose utilizing Pichia galeiformis, was also recovered in the last tanks of the process where the hexose to pentose sugar ratio and the inhibitory pressure are expected to be the lowest.
Saccharomyces cerevisiae; Spent sulfite liquor fermentation; PCR-fingerprinting; Stress tolerance; Resident yeast
Intake of dietary fibres may reduce the prevalence of physiological risk factors of the metabolic syndrome, such as high plasma lipid levels and low-grade inflammatory state. Dietary fibres are usually of plant origin however microbial exopolysaccharides (EPSs) have analogue structures that could potentially exert similar physiological effects. Pediococcus parvulus 2.6 (Pd 2.6) excretes a ropy EPS and has previously shown probiotic potential. The aim of this work was to evaluate physiological effects of Pd 2.6 and its EPS in vivo. The live Pd 2.6 (both the ropy and non-ropy isogenic variant) and its purified EPS were fed to hypercholesterolemic LDL-receptor deficient mice for 6 weeks to investigate their effects on cholesterol levels and the inflammatory tone of the animals. Both variants of Pd 2.6 survived passage through the mouse gut fulfilling an important criterion of probiotics. The ability to produce EPS was conferring an advantage to survival (faecal recovery of 3.7 (1.9-8.7) vs. 0.21 (0.14-0.34) *108 CFU, P < 0.001, median and 25th and 75th percentiles). The ropy Pd 2.6 decreased the levels of soluble vascular cell adhesion molecule-1 compared to the EPS alone (591 ± 14 vs. 646 ± 13 ng/ml, P < 0.05). An increase in liver weight in mice fed the purified EPS was observed, but with no change in liver lipids. No changes in blood lipids were detected in any group. Further the EPS induced growth of the caecal tissue and increased the amount of caecal content showing bulking properties like that of a dietary fibre.
Pediococcus parvulus; Cholesterol; Exopolysaccharide; Lactic acid bacteria
Bacterial resistance against antibiotic treatment has become a major threat to public health. Antimicrobial peptides (AMPs) have emerged as promising alternative agents for treatment of infectious diseases. This study characterizes novel synthetic peptides sequentially derived from the AMP centrocin 1, isolated from the green sea urchin, for their applicability as anti-infective agents.
The microbicidal effect of centrocin 1 heavy chain (CEN1 HC-Br), its debrominated analogue (CEN1 HC), the C-terminal truncated variants of both peptides, i.e. CEN1 HC-Br (1–20) and CEN1 HC (1–20), as well as the cysteine to serine substituted equivalent CEN1 HC (Ser) was evaluated using minimal microbicidal concentration assay. The anti-inflammatory properties were assessed by measuring the inhibition of secretion of pro-inflammatory cytokines. All the peptides tested exhibited marked microbicidal and anti-inflammatory properties. No difference in efficacy was seen comparing CEN1 HC-Br and CEN1 HC, while the brominated variant had higher cytotoxicity. C-terminal truncation of both peptides reduced salt-tolerability of the microbicidal effect as well as anti-inflammatory actions. Also, serine substitution of cysteine residue decreased the microbicidal effect. Thus, from the peptide variants tested, CEN1 HC showed the best efficacy and safety profile. Further, CEN1 HC significantly reduced bacterial counts in two different animal models of infected wounds, while Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) failed to develop resistance against this peptide under continued selection pressure. In summary, CEN1 HC appears a promising new antimicrobial agent, and clinical studies are warranted to evaluate the applicability of this AMP for local treatment of infections in man.
Antibacterial peptide; Wound infection; Antibiotic resistance; MRSA
Agro-industrial wastes are generated during the industrial processing of agricultural products. These wastes are generated in large amounts throughout the year, and are the most abundant renewable resources on earth. Due to the large availability and composition rich in compounds that could be used in other processes, there is a great interest on the reuse of these wastes, both from economical and environmental view points. The economic aspect is based on the fact that such wastes may be used as low-cost raw materials for the production of other value-added compounds, with the expectancy of reducing the production costs. The environmental concern is because most of the agro-industrial wastes contain phenolic compounds and/or other compounds of toxic potential; which may cause deterioration of the environment when the waste is discharged to the nature. Although the production of bioethanol offers many benefits, more research is needed in the aspects like feedstock preparation, fermentation technology modification, etc., to make bioethanol more economically viable.
Bioethanol; Lignocellulosic waste; Immobilization; Mutagenesis; SSF
In this work we performed assays for the genetic improvement of the oleaginous yeast Lipomyces starkeyi DSM 70296 focusing on its utilization for lipid biosynthesis from renewable sources. The genetic optimization was carried out by random mutagenesis by ultraviolet irradiation and mutant selection by cerulenin, a compound displaying inhibitory effects on lipid biosynthesis. Mutants demonstrating normal growth in presence of cerulenin were considered as good candidates for further studies. Using this strategy, we selected 6 mutants for further studies, in which their productivities were evaluated by fermentation in shaken flasks and bioreactor. The evaluation of the fermentative performance of mutants was carried out using xylose as sole carbon source; the fermentation of wild-type strain was used as reference. Using this strategy it was possible to identify one mutant (termed A1) presenting a significant increase in the productivity rates of both biomass and lipid in comparison to wild-type strain. A1 mutant was further studied in bioreactor using the same fermentation parameters optimized for L. starkeyi lipid production from a mixed carbon source (xylose:glucose), as previously determined by other studies in our laboratory. A1 presented a productivity increase of 15.1% in biomass and 30.7% in lipid productivity when compared to the wild-type strain with a similar fatty acid composition, despite a slight increase (approx. 7%) on the unsaturated fraction. Our work demonstrates the feasibility of the random mutagenesis strategy coupled with mutant selection based on cerulenin screening for the genetic improvement of the oleaginous yeast L. starkeyi.
Lipomyces starkeyi; Oleaginous yeast; Random mutagenesis; Cerulenin; Lipids; Biofuels
The G-protein-coupled receptor (GPCR) superfamily, which includes somatostatin receptors (SSTRs), is one of the most important drug targets in the pharmaceutical industry. The yeast Saccharomyces cerevisiae is an attractive host for the ligand screening of human GPCRs. Here, we demonstrate the utility of the technology that was developed for displaying peptide ligands on yeast plasma membrane, termed “PepDisplay”, which triggers signal transduction upon GPCR activation. A yeast strain that heterologously produced human somatostatin receptor subtype-2 (SSTR2) and chimeric Gα protein was constructed along with membrane-displayed somatostatin; somatostatin was displayed on the yeast plasma membrane by linking it to the anchoring domain of the glycosylphosphatidylinositol anchored plasma membrane protein Yps1p. We demonstrate that the somatostatin displayed on the plasma membrane successfully activated human SSTR2 in S. cerevisiae. The methodology presented here provides a new platform for identifying novel peptide ligands for both liganded and orphan mammalian GPCRs.
Membrane-displayed ligand; PepDisplay; Yeast GPCR assay; Cyclic peptide; Somatostatin receptor subtype-2; Chimeric Gα protein
In this study manganese peroxidase (MnP) enzymes from selected white-rot fungi were isolated and compared for potential future recombinant production. White-rot fungi were cultivated in small-scale in liquid media and a simplified process was established for the purification of extracellular enzymes.
Five lignin degrading organisms were selected (Bjerkandera sp., Phanerochaete (P.) chrysosporium, Physisporinus (P.) rivulosus, Phlebia (P.) radiata and Phlebia sp. Nf b19) and studied for MnP production in small-scale. Extracellular MnP activity was followed and cultivations were harvested at proximity of the peak activity. The production of MnPs varied in different organisms but was clearly regulated by inducing liquid media components (Mn2+, veratryl alcohol and malonate). In total 8 different MnP isoforms were purified.
Results of this study reinforce the conception that MnPs from distinct organisms differ substantially in their properties. Production of the extracellular enzyme in general did not reach a substantial level. This further suggests that these native producers are not suitable for industrial scale production of the enzyme. The highest specific activities were observed with MnPs from P. chrysosporium (200 U mg-1), Phlebia sp. Nf b19 (55 U mg-1) and P. rivulosus (89 U mg-1) and these MnPs are considered as the most potential candidates for further studies. The molecular weight of the purified MnPs was estimated to be between 45–50 kDa.
White-rot fungi; Manganese peroxidase; Lignin degradation; Lignocellulose; Enzyme purification
The goal of this study was to investigate the feasibility of bacterial cellulose (BC) scaffold to support osteoblast growth and bone formation. BC was produced by culturing Acetobacter xylinum supplemented with hydroxyapatite (HA) to form BC membranes (without HA) and BC/HA membranes. Membranes were subjected to X-ray photoelectron spectroscopy (XPS) analysis to determine surface element composition. The membranes were further used to evaluate osteoblast growth, alkaline phosphatase activity and bone nodule formation. BC was free of calcium and phosphate. However, XPS analysis revealed the presence of both calcium (10%) and phosphate (10%) at the surface of the BC/HA membrane. Osteoblast culture showed that BC alone was non-toxic and could sustain osteoblast adhesion. Furthermore, osteoblast adhesion and growth were significantly (p ≤0.05) increased on BC/HA membranes as compared to BC alone. Both BC and BC/HA membranes improved osteoconductivity, as confirmed by the level of alkaline phosphatase (ALP) activity that increased from 2.5 mM with BC alone to 5.3 mM with BC/HA. BC/HA membranes also showed greater nodule formation and mineralization than the BC membrane alone. This was confirmed by Alizarin red staining (ARS) and energy dispersive X-ray spectroscopy (EDX). This work demonstrates that both BC and BC/HA may be useful in bone tissue engineering.
Acetobacter xylinum; Hydroxyapatite; Osteoblasts; Tissue regeneration; Cellulose
The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.
16S rRNA gene; Automated nucleic acid extraction platforms; Microbial biofouling; Bacterial community; Oilfield microbiology
Isolation of polyhydroxyalkanoates (PHAs) from bacterial cell matter is a critical step in order to achieve a profitable production of the polymer. Therefore, an extraction method must lead to a high recovery of a pure product at low costs. This study presents a simplified method for large scale poly(3-hydroxybutyrate), poly(3HB), extraction using sodium hypochlorite. Poly(3HB) was extracted from cells of Ralstonia eutropha H16 at almost 96% purity. At different extraction volumes, a maximum recovery rate of 91.32% was obtained. At the largest extraction volume of 50 L, poly(3HB) with an average purity of 93.32% ± 4.62% was extracted with a maximum recovery of 87.03% of the initial poly(3HB) content. This process is easy to handle and requires less efforts than previously described processes.
Poly (3-hydroxybutyrate); Polyhydroxyalkanoates; Ralstonia eutropha; PHA recovery; Sodium hypochlorite; Downstream process
The AcrAB-TolC efflux pump is involved in maintaining intrinsic organic solvent tolerance in Escherichia coli. Mutations in regulatory genes such as marR, soxR, and acrR are known to increase the expression level of the AcrAB-TolC pump. To identify these mutations in organic solvent tolerant E. coli, eight cyclohexane-tolerant E. coli JA300 mutants were isolated and examined by DNA sequencing for mutations in marR, soxR, and acrR. Every mutant carried a mutation in either marR or acrR. Among all mutants, strain CH7 carrying a nonsense mutation in marR (named marR109) and an insertion of IS5 in acrR, exhibited the highest organic solvent-tolerance levels. To clarify the involvement of these mutations in improving organic solvent tolerance, they were introduced into the E. coli JA300 chromosome by site-directed mutagenesis using λ red-mediated homologous recombination. Consequently, JA300 mutants carrying acrR::IS5, marR109, or both were constructed and named JA300 acrRIS, JA300 marR, or JA300 acrRIS marR, respectively. The organic solvent tolerance levels of these mutants were increased in the following order: JA300 < JA300 acrRIS < JA300 marR < JA300 acrRIS marR. JA300 acrRIS marR formed colonies on an agar plate overlaid with cyclohexane and p-xylene (6:4 vol/vol mixture). The organic solvent-tolerance level and AcrAB-TolC efflux pump-expression level in JA300 acrRIS marR were similar to those in CH7. Thus, it was shown that the synergistic effects of mutations in only two regulatory genes, acrR and marR, can significantly increase organic solvent tolerance in E. coli.
Escherichia coli; Organic solvent tolerance; MarR; AcrR; AcrAB-TolC; Efflux pump
S-adenosyl-L-methionine is an important bioactive molecule participating in a number of biochemical reactions including the transmethylation and transsulphuration reactions of proteins and the biosynthesis of aliphatic polyamines. Strategies of metabolic engineering were used to alter the metabolic flux for enhancing the production of S-adenosyl-L-methionine (SAM) in Pichia pastoris GS115. These strategies include the over-expression of Sam2 by knock-in technique and the disruption of Cbs by knock-out technique. Three strains, ZJGSU1 with knock- in of Sam2, ZJGSU2 with knock-out of Cbs and ZJGSU3 with both knock-in of Sam2 and knock -out of Cbs, were constructed for the effective production of SAM. Yields of SAM in strains ZJGSU1 and ZJGSU2 were 32- and 5-fold higher than in the original strain P. pastoris GS115, respectively. The strain ZJGSU3 had a dramatic increase in the SAM yield, and it was 46-fold higher compared to the original strain. These results indicate that there is a strong synergistic effect on the production of SAM by combining knock-in with knock-out techniques. The yield of SAM in ZJGSU3 strain was 4.37 g/L in a 3 L fermentor. This study provides deep insight into the effective industrial production of SAM in future.
S-adenosyl-L-methionine; Pichia pastoris GS115; Metabolic engineering; Fermentation
To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.
Biorefinery; Carbohydrate-binding module (CBM); Cellulase; Yeast cell-surface engineering
Like any other enteric pathogen, Salmonella also encounters acidic stress in the stomach as well as within the host macrophage milieu. However, the pathogen is reported to combat this stress through acid tolerance response (ATR), expressing a number of genes and eventually the proteins. Recently, an acid induced outer membrane phenotype encoded by fliC gene in Salmonella enterica serovar Typhi has been identified. In the present study, fliC gene was cloned to study its biological implications. The recombinant FliC (rFliC) protein was observed to stimulate the production of antibodies. These antibodies could also recognize the FliC protein (antigen) in the clinical samples i.e. blood samples from typhoid patents as well as healthy blood samples spiked with serovar Typhi. Moreover, the rFliC also reacted with the sera from patients suffering with typhoid fever indicating its in-vivo immunogenicity. Ex-vivo study revealed that rFliC has the potential to stimulate the macrophages to generate higher levels of inflammatory mediators such as malondialdehyde (MDA) and nitrite. The inflammatory potential of FliC was also confirmed in-vivo, by the paw oedema test as well as by flicking response of the inflamed paw indicating hyperalgesia occurring during inflammatory response. The findings of the present study indicate that acid induced FliC might be one of the factors enhancing the virulence of serovar Typhi under the host acidic conditions and may prove to be helpful in designing the prophylactic measures.
Outer membrane proteins (OMPs); Salmonella enterica serovar Typhi; Flagellin (FliC); Immunogenicity; Inflammation; Thermal hyperalgesia
The contaminant concentrations over which type strains of the species Dehalogenimonas alkenigignens and Dehalogenimonas lykanthroporepellens were able to reductively dechlorinate 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were evaluated. Although initially isolated from an environment with much lower halogenated solvent concentrations, D. alkenigignens IP3-3T was found to reductively dehalogenate chlorinated alkanes at concentrations comparable to D. lykanthroporepellens BL-DC-9T. Both species dechlorinated 1,2-DCA, 1,2-DCP, and 1,1,2-TCA present at initial concentrations at least as high as 8.7, 4.0, and 3.5 mM, respectively. The ability of Dehalogenimonas spp. to carry out anaerobic reductive dechlorination even in the presence of high concentrations of chlorinated aliphatic alkanes has important implications for remediation of contaminated soil and groundwater.
Bioremediation; Chlorinated alkanes; Dehalogenimonas; Reductive dechlorination; Dehalogenation
Enterococcus faecium No. 78 (PNCM-BIOTECH 10375) isolated from puto, a type of fermented rice in the Philippines was used to produce lactic acid in repeated batch fermentation mode. Enzymatically liquefied sago starch was used as the sole carbon source, since sago (Metroxylon spp) is a sustainable crop for industrial exploitation. Liquefied sago starch was inoculated with E. faecium to perform the saccharification and fermentation processes simultaneously. Results demonstrated that E. faecium was reused for 11 fermentation cycles with an average lactic acid yield of 36.3 ± 4.71 g/l. The lactic acid production was superior to that of simple batch mode and continuous fermentation in terms of lactic acid concentration. An un-dissociated lactic acid concentration of 1.15 mM affected the productivity of the cells. Work is in progress to maintain and increase the usability of the cells over higher fermentation cycles.
Enteroccus faecium; Lactic acid; Repeated batch fermentation; Liquefied sago starch; Cell reuse
Through metabolic engineering microorganisms can be engineered to produce new products and further produce these with higher yield and productivities. Here, we expressed the bacterial polyhydroxybutyrate (PHB) pathway in the yeast Saccharomyces cerevisiae and we further evaluated the effect of engineering the formation of acetyl coenzyme A (acetyl-CoA), an intermediate of the central carbon metabolism and precursor of the PHB pathway, on heterologous PHB production by yeast. We engineered the acetyl-CoA metabolism by co-transformation of a plasmid containing genes for native S. cerevisiae alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA acetyltransferase (ERG10) and a Salmonella enterica acetyl-CoA synthetase variant (acsL641P), resulting in acetoacetyl-CoA overproduction, together with a plasmid containing the PHB pathway genes coding for acetyl-CoA acetyltransferase (phaA), NADPH-linked acetoacetyl-CoA reductase (phaB) and poly(3-hydroxybutyrate) polymerase (phaC) from Ralstonia eutropha H16. Introduction of the acetyl-CoA plasmid together with the PHB plasmid, improved the productivity of PHB more than 16 times compared to the reference strain used in this study, as well as it reduced the specific product formation of side products.
Polyhydroxybutyrate; Acetyl coenzyme A; Saccharomyces cerevisiae; Pathway engineering
A method for the detection of haloalkane conversion to the corresponding alcohols by haloalkane dehalogenases is described. It is based on a multistage enzyme reaction which allows for the analysis of alkyl halides in buffered systems. Irreversible hydrolytic dehalogenation catalyzed by haloalkane dehalogenase DhaA from Rhodococcus erythropolis transfers an alkyl halide into a corresponding alcohol that is further oxidized by alcohol oxidase AOX from Pichia pastoris yielding a respective aldehyde and hydrogen peroxide easily detectable via the horseradish peroxidase catalyzed oxidation of chromogenic molecules. Due to its high sensitivity (0.025 mM, 0.43 ppm for 1,3-dibromopropane), low expenditure and the ability of handling a large number of samples in parallel, this method is an attractive alternative to existing procedures for the monitoring of both haloalkanes and dehalogenases.
Alcohol oxidase; Haloalkane dehalogenase; Haloalkanes; Horseradish peroxidase; Multistage enzyme reaction
We recently established a method for isolating functional single cells from environmental samples using a micromanipulator (Functional single-cell (FSC) isolation), and applied it to the study of denitrifying bacteria in rice paddy soil (Ashida et al. 2010. Appl Microbiol Biotechnol 85:1211–1217). To further examine the advantages and possible disadvantages of the FSC method, we isolated denitrifying bacteria from the same rice paddy soil sample using both FSC and standard agar plate dilution (APD) methods and compared in this study. The proportion of denitrifying bacteria in the total isolates was more than 6-fold larger with FSC isolation (57.1%) compared with the APD method (9.2%). Denitrifying bacteria belonging to Alphaproteobacteria and Bacilli were commonly isolated using both methods, whereas those belonging to Betaproteobacteria, which had been found to be active in the denitrification-inductive paddy soil, were isolated only with the FSC method. On the other hand, Actinobacteria were only isolated using the APD method. The mean potential denitrification activity of the FSC isolates was higher than that of the APD isolates. Overall, FSC isolation was confirmed to be an excellent method for studying denitrifying bacteria compared with the standard agar plate dilution method.
16S rRNA gene; Denitrifying bacteria; Functional single-cell isolation; Phylogenetic analysis; Rice paddy soil
Fusarium proliferatum NBRC109045 is a filamentous fungus isolated from Vietnamese forest due to high production of β-glucosidases. Production of the enzyme was studied on varied carbon source based mediums. The highest activity was obtained in medium containing 1% corn stover + 1% wheat bran (3.31 ± 0.14 U/ml). It is interesting to note that glucose (0.69 ± 0.02 U/ml) gave higher activity and just followed by cellobiose among the di- and mono-saccharides, which is generally regarded as a universal repressor of hydrolases. We improved the zymogram method to prove that in response to various carbon sources, F. proliferatum could express various β-glucosidases. One of the β-glucosidases produced by F. proliferatum growing in corn stover + wheat bran based medium was partially purified and proved to have high catalytic ability.
Fusarium proliferatum; β-glucosidases; Differential expression; The translation elongation factor 1-α
The biopreservation of foods using bacteriocinogenic lactic acid bacteria (LAB) isolated directly from foods is an innovative approach. The objectives of this study were to isolate and identify bacteriocinogenic LAB from various cheeses and yogurts and evaluate their antimicrobial effects on selected spoilage and pathogenic microorganisms in vitro as well as on a food commodity.
LAB were isolated using MRS and M17 media. The agar diffusion bioassay was used to screen for bacteriocin or bacteriocin-like substances (BLS) producing LAB using Lactobacillus sakei and Listeria innocua as indicator organisms. Out of 138 LAB isolates, 28 were found to inhibit these bacteria and were identified as strains of Enterococcus faecium, Streptococcus thermophilus, Lactobacillus casei and Lactobacillus sakei subsp. sakei using 16S rRNA gene sequencing. Eight isolates were tested for antimicrobial activity at 5°C and 20°C against L. innocua, Escherichia coli, Bacillus cereus, Pseudomonas fluorescens, Erwinia carotovora, and Leuconostoc mesenteroides subsp. mesenteroides using the agar diffusion bioassay, and also against Penicillium expansum, Botrytis cinerea and Monilinia frucitcola using the microdilution plate method. The effect of selected LAB strains on L. innocua inoculated onto fresh-cut onions was also investigated.
Twenty percent of our isolates produced BLS inhibiting the growth of L. innocua and/or Lact. sakei. Organic acids and/or H2O2 produced by LAB and not the BLS had strong antimicrobial effects on all microorganisms tested with the exception of E. coli. Ent. faecium, Strep. thermophilus and Lact. casei effectively inhibited the growth of natural microflora and L. innocua inoculated onto fresh-cut onions. Bacteriocinogenic LAB present in cheeses and yogurts may have potential to be used as biopreservatives in foods.
Lactic acid bacteria; Bacteriocins; Bacteriocin-like substance (BLS); Antimicrobials; Fresh-cut onions
Here, we determined the potential of photochemotherapy, namely the application of photodynamic compounds followed by exposure to a suitable source of UV-visible radiation against corneal pathogen, Acanthamoeba. Organometallic macromolecule, tin porphyrin [Sn(IV)porphyrin] was synthesized and purity confirmed using nuclear magnetic resonance spectroscopy. The Sn(IV)porphyrin was tested against a keratitis isolate of Acanthamoeba castellanii belonging to the T4 genotype using growth and viability assays. The effects of Sn(IV)porphyrin on A. castellanii binding to and cytopathogenicity of human corneal epithelial cells in vitro were tested. The metalloporphyrin showed potent amoebistatic effects. The tin porphyrin inhibited amoebae binding to and cytopathogenicity of corneal epithelial cells. By using derivatives of photodynamic compounds [Sn(IV)porphyrin-antibody conjugates] for selective targeting of the parasite together with appropriate selection of light source will determine the potential of photochemotherapy against Acanthamoeba keratitis.
Acanthamoeba; Keratitis; Tin porphyrin; Treatment; Drug resistance; NMR
The production of ethanol has been considered as an alternative to replace part of the petroleum derivate. Brazil and the US are the leading producers, but more environmentally friendly alternatives are needed. Lignocellulose has an enormous potential but technology has to be still improve in order to economically produce ethanol. The present paper reviews the potential and problems of this technology and proposes the study of a group of microorganisms with the largest genetic pool, marine microorganism.
Isopropanol represents a widely-used commercial alcohol which is currently produced from petroleum. In nature, isopropanol is excreted by some strains of Clostridium beijerinckii, simultaneously with butanol and ethanol during the isopropanol butanol ethanol (IBE) fermentation. In order to increase isopropanol production, the gene encoding the secondary-alcohol dehydrogenase enzyme from C. beijerinckii NRRL B593 (adh) which catalyzes the reduction of acetone to isopropanol, was cloned into the acetone, butanol and ethanol (ABE)-producing strain C. acetobutylicum ATCC 824. The transformants showed high capacity for conversion of acetone into isopropanol (> 95%). To increase isopropanol production levels in ATCC 824, polycistronic transcription units containing, in addition to the adh gene, homologous genes of the acetoacetate decarboxylase (adc), and/or the acetoacetyl-CoA:acetate/butyrate:CoA transferase subunits A and B (ctfA and ctfB) were constructed and introduced into the wild-type strain. Combined overexpression of the ctfA and ctfB genes resulted in enhanced solvent production. In non-pH-controlled batch cultures, the total solvents excreted by the transformant overexpressing the adh, ctfA, ctfB and adc genes were 24.4 g/L IBE (including 8.8 g/L isopropanol), while the control strain harbouring an empty plasmid produced only 20.2 g/L ABE (including 7.6 g/L acetone). The overexpression of the adc gene had limited effect on IBE production. Interestingly, all transformants with the adh gene converted acetoin (a minor fermentation product) into 2,3-butanediol, highlighting the wide metabolic versatility of solvent-producing Clostridia.
Clostridium; Butanol; Biobutanol; Isopropanol; IBE fermentation; 2,3-butanediol; Acyl-CoA transferase