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Applied Microbiology and Biotechnology (v.55, #1)
Microbial formation, biotechnological production and applications of 1,2-propanediol by G. N. Bennett; K.-Y. San (pp. 1-9).
This short review covers metabolic pathways, genetics and metabolic engineering of 1,2-propanediol formation in microbes. 1,2-Propanediol production by bacteria and yeasts has been known for many years and two general pathways are recognized. One involves the metabolism of deoxyhexoses, where lactaldehyde is formed during the glycolytic reactions and is then reduced to 1,2-propanediol. The second pathway derives from the formation of methylglyoxal from dihydroxyacetonephosphate and its subsequent reduction to 1,2-propanediol. The enzymes involved in the reduction of methylglyoxal can generate isomers of lactaldehyde or acetol, which can be further reduced by specific reductases, giving chiral 1,2-propanediol as the product. The stereospecificity of the enzymes catalyzing the two reduction steps is important in deriving a complete pathway. Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose. The optimization of these strains may yield microbial processes for the production of this widely used chemical.
Biological production of 2,3-butanediol by M.-J. Syu (pp. 10-18).
2,3-Butanediol (2,3-BDL), which is very important for a variety of chemical feedstocks and liquid fuels, can be derived from the bioconversion of natural resources. One of its well known applications is the formation of methyl ethyl ketone, by dehydration, which can be used as a liquid fuel additive. This article briefly reviews the basic properties of 2,3-BDL and the metabolic pathway for the microbial formation of 2,3-BDL. Both the biological production of 2,3-BDL and the variety of strains being used are introduced. Genetically improved strains for BDL production which follow either the original mechanisms or new mechanisms are also described. Studies on fermentation conditions are briefly reviewed. On-line analysis, modeling, and control of BDL fermentation are discussed. In addition, downstream recovery of 2,3-BDL and the integrated process (being important issues of BDL production) are also introduced.
Pro- and prebiotics – the tasty guardian angels? by Rainer Simmering; Michael Blaut (pp. 19-28).
It is generally accepted that the bacterial community resident in the human intestinal tract has a major impact on gastrointestinal function and thereby on human health and well-being. Considerable efforts have been made to influence the intestinal microbiota by dietary means in such a way that the health of the host is beneficially affected. Pro- and prebiotics are food products that are specially designed for this purpose. Parallel to the increase in the acceptance of such products by the consumer, the scientific interest in the mechanisms underlying their presumed effects, such as pathogen inhibition, immune modulation or anti-carcinogenicity, has grown continuously in recent years. Some of these effects have been established by several independent studies, but others are still controversial. This review relates the health claims made for the pro- and prebiotic food products to the facts established by in vivo and in vitro studies. The assessment of pro- and prebiotic effects on the microbial gut ecosystem is highly improved and facilitated by the application of molecular methods. Biotechnological aspects of the production of pro- and prebiotics are discussed.
Anaerobic sequencing batch reactors for wastewater treatment: a developing technology by M. Zaiat; J. A. D. Rodrigues; S. M. Ratusznei; E. F. M. de Camargo; W. Borzani (pp. 29-35).
This paper describes and discusses the main problems related to anaerobic batch and fed-batch processes for wastewater treatment. A critical analysis of the literature evaluated the industrial application viability and proposed alternatives to improve operation and control of this system. Two approaches were presented in order to make this anaerobic discontinuous process feasible for industrial application: (1) optimization of the operating procedures in reactors containing self-immobilized sludge as granules, and (2) design of bioreactors with inert support media for biomass immobilization.
Chemostat study of xylitol production by Candida guilliermondii by T. Granström; H. Ojamo; M. Leisola (pp. 36-42).
The mechanism of production of xylitol from xylose by Candida guilliermondii was studied using chemostat cultures and enzymatic assays. The maximum dilution rate in aerobic conditions was 0.34 1/h. No xylitol was produced. Under oxygen-limited conditions xylose uptake was impaired and glycerol accumulated but no xylitol was detected. Under transient oxygen limitation, caused by a gradual decrease in the agitation rate, onset of xylitol, acetate and residual xylose accumulation occurred simultaneously when q O2 dropped below 25 mmol/C-mmol cell dry weight (CDW) per hour. Ethanol and glycerol started to accumulate when q O2 dropped below 20 mmol/C-mmol CDW per hour. The highest in vitro enzyme activities were found at the lowest dilution rate studied (0.091/h) under aerobic conditions. The amount of active enzymes or cofactor availability did not limit the rate of xylose consumption. Our results confirm that a surplus of NADH during transient oxygen limitation inhibited the activity of xylitol dehydrogenase which resulted in xylitol accumulation. Phosphoglucoisomerase (E.C. 5.3.1.9.) and glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) activities suggest re-shuttling of the metabolites into the pentose phosphate pathway.
Anaerobic desulfurization of ground rubber with the thermophilic archaeon Pyrococcus furiosus – a new method for rubber recycling by Katarina Bredberg; Jonas Persson; Magdalena Christiansson; Bengt Stenberg; Olle Holst (pp. 43-48).
The anaerobic sulfur-reducing archaeon Pyrococcus furiosus was investigated regarding its capacity to desulfurize rubber material. The microorganism's sensitivity towards common rubber elastomers and additives was tested and several were shown to be toxic to P. furiosus. The microorganism was shown to utilize sulfur in vulcanized natural rubber and an increase in cell density was obtained when cultivated in the presence of spent tire rubber. Ethanol-leached cryo-ground tire rubber treated with P. furiosus for 10 days was vulcanized together with virgin rubber material (15% w/w) and the mechanical properties of the resulting material were determined. The increase in the stress at break value and the decrease in swell ratio and stress relaxation rate obtained for material containing microbially treated rubber (compared to untreated material) show the positive effects of microbial desulfurization on rubber.
Idle time in the washing and iron concentration in leachate removed: two basic parameters in the desulphurization of coal in a packed column by J. Cara; A. Aller; M. Otero; A. Morán (pp. 49-54).
Column biodesulphurization of coal is at the experimental stage and is influenced by many variables including temperature, pH, particle size, concentration of iron in solution, among others. Idle time in the washing process and the concentration of dissolved iron in the purged leachate are two variables with a definite effect on the yield of the desulphurization system. In the laboratory, several trials were run with columns packed with coal for different idle times: 1, 2, 3, 5, 6 and 7 days, and for different concentrations of iron in the purged leachate: 500 to 4,000 mg/l. The optimal values for the two variables; that is, those allowing for the highest desulphurization yields, were idle times of 3 and 5 days, which give an elimination of 56% and 49% of pyritic sulphur, respectively, and 3,000 mg/l of iron concentration in the purged leachate, giving a decrease in pyritic sulphur in coal of 57%.
Application of high performance anion exchange chromatography to study invertase-catalysed hydrolysis of sucrose and formation of intermediate fructan products by S. Farine; C. Versluis; P. Bonnici; A. Heck; C. L'homme; A. Puigserver; A. Biagini (pp. 55-60).
Baker's yeast invertase was found to catalyse transfructosylation reactions in aqueous and anhydrous organic media with sucrose as a substrate, leading to the formation of five intermediate fructans in addition to the release of D-glucose (D-Glc)and D-fructose (D-Fru). All the reaction products were separated and quantitatively estimated using high performance anion exchange-pulsed amperometric detection equipment. The unknown products were subsequently identified by linkage analysis as β-D-Fru-(2→1)-β-D-Fru-(2→1)-α-D-glucopyranoside (1-kestose), β-D-Fru-(2→6)-α-D-glucopyranoside (6-β-fructofuranosylglucose), β-D-Fru-(2→1)-β-D-fructofuranoside (inulobiose), β-D-Fru-(2→6)-β-D-Fru-(2→1)-α-D-glucopyranoside (6-kestose) and β-D-Fru-(2→6)-α-D-Glc-(1→2)-β-D-fructofuranoside (neokestose); and this last was eluted together with a disaccharide. The time-course of sucrose hydrolysis via fructan production in 2 ml of a 50 mM sodium acetate buffer (pH 4.5) containing 0.2 M sucrose and 25 U of invertase was different from that in 2 ml of anhydrous toluene with 1.46 M sucrose and 1,000 U of invertase as a suspended powder. Under the latter experimental conditions, invertase was found to exhibit cyclic behaviour, where sucrose was degraded and subsequently synthesised. This observation has not yet been reported, as far as we know.
Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining by F. I. J. Pastor; X. Pujol; A. Blanco; T. Vidal; A. L. Torres; P. Díaz (pp. 61-68).
The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known β-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca2+ and showed its maximum at 53 °C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect.
Construction of protein overproducer strains in Bacillus subtilis by an integrative approach by J. Jan; F. Valle; F. Bolivar; E. Merino (pp. 69-75).
We evaluated the effect of several genetic factors reported as having a role in the induction of the expression of significant levels of recombinant protein in Bacillus subtilis. We utilized the β-galactosidase reporter protein from Escherichia coli as our model for measuring the overproduction of heterologous proteins in B. subtilis. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. In this study, we considered factors known to modulate the transcription and translation initiation rates and genetic and mRNA stability. We also consider the effects of different genetic backgrounds, such as degU32 and hpr2, that until now have been studied independently. By changing the native −35 promoter box to the consensus TTGACA sequence of the aprE promoter, a significant 100-fold increase in the β-galactosidase activity was obtained. On the other hand, changes such as the GTG to ATG start codon, the construction of a consensus AAGGAGG ribosome binding site, and the addition of the cryIIIA transcription terminator at the 3′ end of the lacZ gene, produced only marginal effects on the final β-galactosidase activity.
Xylitol production by recombinant Saccharomyces cerevisiae expressing the Pichia stipitis and Candida shehatae XYL1 genes by R. Govinden; B. Pillay; W. H. van Zyl; D. Pillay (pp. 76-80).
The xylose reductase gene (XYL1) was isolated from Pichia stipitis and Candida shehatae, cloned into YEp-based vectors under the control of ADH2 and PGK1 promoter/terminator cassettes and introduced into Saccharomyces cerevisiae Y294 by electroporation. Shake-flask fermentations were carried out with 5% xylose and 1% galactose, glucose or maltose as co-substrates. Xylose uptake was similar in both the recombinant strains when different co-substrates were used and slowed once the co-substrate was depleted. The recombinant strains converted xylose to xylitol with yields approaching the theoretical maxima. Xylitol production was most rapid when the co-substrate was still present. Approximately 50% of the xylose was not metabolized due to the depletion of the co-substrate.
α-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans by T. Agger; A. B. Spohr; J. Nielsen (pp. 81-84).
The effect of biomass concentration on the formation of Aspergillus oryzaeα-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of α-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2–12 g dry weight kg−1. When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzaeα-amylase gene (creA) was deleted, no significant decrease in the specific rate of α-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific α-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium.
Carrier-mediated transport of riboflavin in Ashbya gossypii by ; ; (pp. 85-89).
The filamentous hemiascomycete Ashbya gossypii is used for industrial riboflavin production. We examined riboflavin uptake and excretion at the plasma membrane using riboflavin auxotrophic and overproducing mutants. The riboflavin uptake system had low activity [V max=20±4 nmol min–1 g–1 mycelial dry weight (dw)] and high affinity (K M=40±12 µM). Inhibitor studies with the analogs FMN and FAD revealed high specificity of the uptake system. Excretion of riboflavin was not the consequence of non-specific permeability of the plasma membrane. Excretion rates in the mid-production phase were determined to be 2.5 nmol min–1 g–1 dw for wild-type cells and 66.7 nmol min–1 g–1 dw for an overproducing mutant, respectively. Inhibition of the reverse reaction, riboflavin uptake, led to an increase in apparent riboflavin efflux in the early production phase, indicating the presence of a separate excretion carrier. Riboflavin accumulation in A. gossypii vacuoles leading to product retention was found to be a secondary transport process. To address the question of whether a flux from the vacuoles back into the cytoplasm is present, we characterized efflux in hyphae in which the plasma membrane was permeabilized with digitonin. Efflux kinetics across the vacuolar membrane were unaffected by the lack of vacuolar H+-ATPase activity and ATP, suggesting a passive mechanism. Based on the characterization of riboflavin transport processes in this study, the design of new production strains with improved riboflavin excretion may be possible.
Nitrate reduction by Citrobacter diversus under aerobic environment by H. K. Huang; S. K. Tseng (pp. 90-94).
A new aerobic denitrifier, Citrobacter diversus, was isolated from both nitrification and denitrification sludge. To monitor the variation in the concentration of nitrogen oxides, aerobic denitrification by C. diversus was carried out in a batch reactor. When the nitrate concentration was greater than 180 mg N l−1, the nitrate reduction rate became stable. The effect of the C/N ratio on the denitrification activity was also investigated. The results showed that the optimum denitrification activity was obtained when the C/N ratio was 4–5. The range of the C/N ratio was higher than that for traditional anoxic denitrification. The effect of the dissolved oxygen concentration was further studied; and it was found that the range of dissolved oxygen concentrations, both for specific growth rates and for specific denitrification rates, was 2–6 mg−1. From these results, it can be concluded that both the concentration of dissolved oxygen and the C/N ratio are key factors in the aerobic denitrification by C. diversus.
Enzymatic reduction of chromate: comparative studies using sulfate-reducing bacteria by C. Michel; M. Brugna; C. Aubert; A. Bernadac; M. Bruschi (pp. 95-100).
Various sulfate-reducing bacteria of the genera Desulfovibrio and Desulfomicrobium were tested and compared for enzymatic reduction of chromate. Our study demonstrated that the ability to reduce chromate is widespread among sulfate-reducing bacteria. Among them, Desulfomicrobium norvegicum reduced Cr(VI) with the highest reaction rate. This strain grew in the presence of up to 500 μM chromate, but Cr(VI) reduction in the absence of sulfate was not associated with growth. The presence of chromate induced morphological changes and leakage of periplasmic proteins into the medium. The ability of isolated polyheme cytochromes c from sulfate- and sulfur-reducing bacteria to reduce chromate was also analyzed. Tetraheme cytochrome c 3(M r. 13,000) from Desulfomicrobium norvegicum showed twice as much activity as either tetraheme cytochrome c 3 from Desulfovibrio vulgaris strain Hildenborough or triheme cytochrome c 7 from Desulfuromonas acetoxidans. Results with cytochromes c 3 and other c-type cytochromes altered by site-directed mutagenesis indicated that negative redox potential hemes are crucial for metal reductase activity. The present study also demonstrated that the (Fe) hydrogenase from sulfate-reducing bacteria could reduce chromate.
Biodegradation of sulfanilic acid by Pseudomonas paucimobilis by K. Perei; G. Rákhely; I. Kiss; B. Polyák; K. L. Kovács (pp. 101-107).
An aerobic bacterium, isolated from a contaminated site, was able to degrade sulfanilic acid (4-aminobenzenesulfonic acid) and was identified as Pseudomonas paucimobilis. The isolate could grow on sulfanilic acid (SA) as its sole carbon and nitrogen source and metabolized the target compound to biomass. The bioconversion capacity depended on the sulfanilic acid concentration; greater than 98% elimination of the hazardous compound was achieved at low (10 mM) sulfanilic acid concentration, and the yield was greater than 70% at 50 mM concentration of the contaminant. The maximum conversion rate was 1.5 mmol sulfanilic acid/h per mg wet cells at 30 °C. Ca-alginate-phytagel proved a good matrix for immobilization of P. paucimobilis, with essentially unaltered biodegradation activity. Removal of sulfanilic acid from contaminated industrial waste water was demonstrated. SDS-PAGE analysis of the crude extract revealed novel proteins appearing upon induction with sulfanilic acid and related compounds, which indicated alternative degradation mechanisms involving various inducible enzymes.
Genetic reidentification of the pectinolytic yeast strain SCPP as Saccharomyces bayanus var. uvarum by G. I. Naumov; E. S. Naumova; M. Aigle; I. Masneuf; A. Belarbi (pp. 108-111).
Using genetic hybridization analysis, pulsed-field gel electrophoresis of chromosomal DNA and PCR/RFLP analysis of the MET2 gene, we reidentified 11 Champagne yeast strains. Two of them, SCPP and SC4, were found to belong to Saccharomyces bayanus var. uvarum and the remaining strains to S. cerevisiae. Strain SCPP (CLIB 2025) of S. bayanus var. uvarum is known as a producer of three pectinolytic enzymes.
Development and utilisation of a medium to isolate phenanthrene-degrading Pseudomonas spp. by S. Andersen; C. Jørgensen; C. Jacobsen (pp. 112-116).
In this study, we isolated phenanthrene degraders belonging to Pseudomonas spp. by combining the selective force of two previously described media. The two compounds, sodium lauryl sarcosine and trimethoprim, from the Gould S1 medium, were added to minimal agar plates sprayed with phenanthrene. Pseudomonas spp. that could produce clearing zones were isolated in one step from the rhizosphere without first selecting for Pseudomonas spp. and subsequently screening for degraders or vice versa. Enumeration and isolation of Pseudomonas spp. attached to the rhizosphere showed clear differences between two types of soil. Rhizosphere-attached phenanthrene degraders (from Pseudomonas spp.) were isolated from a former coal gasification site, but were absent in an agricultural soil subjected to organic farming. We isolated 23 phenanthrene degraders producing clearing zones from the rhizosphere of barley roots. All of these 23 isolates (of which 16 were fluorescent in UV light) proved to be members of the Pseudomonas RNA homology group I, on the basis of results of the analytical profile index (API) test system and classic taxonomic tests.
Biodegradation of ethyl t-butyl ether (ETBE), methyl t-butyl ether (MTBE) and t-amyl methyl ether (TAME) by Gordonia terrae by Guillermina Hernandez-Perez; Françoise Fayolle; Jean-Paul Vandecasteele (pp. 117-121).
Gordonia terrae strain IFP 2001 was selected from activated sludge for its capacity to grow on ethyl t-butyl ether (ETBE) as sole carbon and energy source. ETBE was stoichiometrically degraded to t-butyl alcohol (TBA) and the activity was inducible. A constitutive strain, G. terrae IFP 2007, derived from strain IFP 2001, was also selected. Methyl t-butyl ether (MTBE) and t-amyl methyl ether (TAME) were not used as carbon and energy sources by the two strains, but cometabolic degradation of MTBE and TAME was demonstrated, to TBA and t-amyl alcohol (TAA) respectively, in the presence of a carbon source such as ethanol. No two-carbon compound was detected during growth on ETBE, but formate was produced during cometabolic degradation of MTBE or TAME. A monooxygenase was involved in the degradation of ethers, because no degradation of ETBE was observed under anaerobic conditions and the presence of a cytochrome P-450 was demonstrated in G. terrae IFP 2001 after induction by cultivation on ETBE.
BTEX catabolism interactions in a toluene-acclimatized biofilter by C. A. du Plessis; J. M. Strauss; K.-H. J. Riedel (pp. 122-128).
BTEX substrate interactions for a toluene-acclimatized biofilter consortium were investigated. Benzene, ethylbenzene, o-xylene, m-xylene and p-xylene removal efficiencies were determined at a loading rate of 18.07 g m−3 h−1 and retention times of 0.5–3.0 min. This was also repeated for toluene in a 1:1 (m/m) ratio mixture (toluene: benzene, ethylbenzene, or xylene ) with each of the other compounds individually to obtain a final total loading of 18.07 g m−3 h−1. The results obtained were modelled using Michaelis–Menten kinetics and an explicit finite difference scheme to generate v max and K m parameters. The v max/K m ratio (a measure of the catalytic efficiency, or biodegradation capacity, of the reactor) was used to quantify substrate interactions occurring within the biofilter reactor without the need for free-cell suspended and monoculture experimentation. Toluene was found to enhance the catalytic efficiency of the reactor for p-xylene, while catabolism of all the other compounds was inhibited competitively by the presence of toluene. The toluene-acclimatized biofilter was also able to degrade all of the other BTEX compounds, even in the absence of toluene. The catalytic efficiency of the reactor for compounds other than toluene was in the order: ethylbenzene>benzene>o-xylene>m-xylene>p-xylene. The catalytic efficiency for toluene was reduced by the presence of all other tested BTEX compounds, with the greatest inhibitory effect being caused by the presence of benzene, while o-xylene and p-xylene caused the least inhibitory effect. This work illustrated that substrate interactions can be determined directly from biofilter reactor results without the need for free-cell and monoculture experimentation.