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Applied Microbiology and Biotechnology (v.60, #1-2)
Metabolic engineering towards biotechnological production of carotenoids in microorganisms by P. Lee; C. Schmidt-Dannert (pp. 1-11).
Carotenoids are important natural pigments produced by many microorganisms and plants. Traditionally, carotenoids have been used in the feed, food and nutraceutical industries. The recent discoveries of health-related beneficial properties attributed to carotenoids have spurred great interest in the production of structurally diverse carotenoids for pharmaceutical applications. The availability of a considerable number of microbial and plant carotenoid genes that can be functionally expressed in heterologous hosts has opened ways for the production of diverse carotenoid compounds in heterologous systems. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms for improved carotenoid productivity. In addition, we will discuss the application of combinatorial and evolutionary strategies to carotenoid pathway engineering to broaden the diversity of carotenoid structures synthesized in recombinant hosts.
Bacterial cysteine desulfurases: their function and mechanisms by H. Mihara; N. Esaki (pp. 12-23).
Cysteine desulfurase is a pyridoxal 5′-phosphate (PLP)-dependent homodimeric enzyme that catalyzes the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. Increased evidence for the functions of cysteine desulfurases has revealed their important roles in the biosyntheses of Fe-S clusters, thiamine, thionucleosides in tRNA, biotin, lipoic acid, molybdopterin, and NAD. The enzymes are also proposed to be involved in cellular iron homeostasis and in the biosynthesis of selenoproteins. The mechanisms for sulfur mobilization mediated by cysteine desulfurases are as yet unknown, but enzymes capable of providing a variety of biosynthetic pathways for sulfur/selenium-containing biomolecules are probably applicable to the production of cofactors and the bioconversion of useful compounds.
Anaerobic sugar catabolism in Lactococcus lactis: genetic regulation and enzyme control over pathway flux by Muriel Cocaign-Bousquet; Sergine Even; Nicolas D. Lindley; Pascal Loubière (pp. 24-32).
Lactic acid bacteria and particularly Lactococcus lactis are widely used for the production of lactic acid in fermented foods. Control of the catabolic rate in L. lactis, i.e., the rate of lactic acid production, appears to be determinant for dairy product quality. While the mechanisms involved in control have not been totally elucidated, they seem to depend upon the strain and the growth conditions. Furthermore, it remains unclear whether the catabolic rate is controlled at the level of transcription, translation or enzyme activity. The recent sequencing of the L. lactis genome has brought novel insights to physiologic studies of the bacteria. This review discusses both genetic information and metabolic studies concerning anaerobic sugar catabolism in L. lactis.
The nitrile-degrading enzymes: current status and future prospects by A. Banerjee; R. Sharma; U. Banerjee (pp. 33-44).
Nitrile-converting enzymes are becoming commonplace in the synthesis of pharmaceuticals and commodity chemicals. These versatile biocatalysts have potential applications in different fields including synthetic biocatalysis and bioremediation. This review attempts to describe in detail the three major classes of nitrile-converting enzymes, namely nitrilases, nitrile hydratases and amidases. Various aspects of these enzymes including their occurrence, mechanism of action, characteristics and applicability in different sectors have been elaborately elucidated. Cloning of genes related to nitrile-converting enzymes is also discussed.
Recent developments in molecular techniques for identification and monitoring of xenobiotic-degrading bacteria and their catabolic genes in bioremediation by J. Widada; H. Nojiri; T. Omori (pp. 45-59).
The pollution of soil and water with xenobiotics is widespread in the environment and is creating major health problems. The utilization of microorganisms to clean up xenobiotics from a polluted environment represents a potential solution to such environmental problems. Recent developments in molecular-biology-based techniques have led to rapid and accurate strategies for monitoring, discovery and identification of novel bacteria and their catabolic genes involved in the degradation of xenobiotics. Application of these techniques to bioremediation has also improved our understanding of the composition, phylogeny, and physiology of metabolically active members of the microbial community in the environment. This review provides an overview of recent developments in molecular-biology-based techniques and their application in bioremediation of xenobiotics.
Study of two-stage processes for the microbial production of 1,3-propanediol from glucose by M. Hartlep; W. Hussmann; N. Prayitno; I. Meynial-Salles; A.-P. Zeng (pp. 60-66).
The microbial production of 1,3-propanediol (1,3-PD) from glucose was studied in a two-stage fermentation process on a laboratory scale. In the first stage, glucose was converted to glycerol either by the osmotolerant yeast Pichia farinosa or by a recombinant Escherichia coli strain. In the second stage, glycerol in the broth from the first stage was converted to 1,3-PD by Klebsiella pneumoniae. The culture broth from P. farinosa was shown to contain toxic metabolites that strongly impair the growth of K. pneumoniae and the formation of 1,3-PD. Recombinant E. coli is more suitable than P. farinosa for producing glycerol in the first stage. The fermentation pattern from glycerol can be significantly altered by the presence of acetate, leading to a significant reduction of PD yield in the second stage. However, in the recombinant E. coli culture acetate formation can be prevented by fed-batch cultivation under limiting glucose supply, resulting in an effective production of 1,3-PD in the second stage with a productivity of 2.0 g l–1 h–1 and a high yield (0.53 g/g) close to that of glycerol fermentation in a synthetic medium. The overall 1,3-PD yield from glucose in the two stage-process with E. coli and K. pneumoniae reached 0.17 g/g.
Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process by S. Alfenore; C. Molina-Jouve; S. Guillouet; J.-L. Uribelarrea; G. Goma; L. Benbadis (pp. 67-72).
Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30°C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l–1 to 135 g l–1 and 147 g l–1, respectively. A maximum instantaneous productivity of 9.5 g l–1 h–1 was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.
Production and stability of 3-hydroxypropionaldehyde in Lactobacillus reuteri by Q. Lüthi-Peng; S. Schärer; Z. Puhan (pp. 73-80).
3-Hydroxypropionaldehyde (3-HPA) is considered as a potent antimicrobial substance. Exploration of its application as a food preservative or as a therapeutic auxiliary agent has been documented in the literature. In the present work, factors that may impact on 3-HPA accumulation by Lactobacillus reuteri and on the stability of 3-HPA were investigated. Three media – H2O, milk and MRS broth – were chosen as test systems. Data indicated that 3-HPA accumulation in resting cells of L. reuteri in a two-step fermentation is greatly affected by temperature, pH, cell age and biomass as well as components in the test system. Within 2 h of incubation, 170 mM 3-HPA could be produced with a cell dry weight of 30 g/l, representing 85% of the glycerol supplied (200 mM) in H2O. The presence of glycerol during cell growth increased the productivity of 3-HPA by resting cells. In general, 3-HPA is much more stable in H2O than in milk and MRS. Factors that enhanced accumulation of 3-HPA did not simply show the same positive impact on the stability of 3-HPA. Thus, for defined applications, factors affecting production and stability of 3-HPA should be evaluated separately.
Citric acid production by Candida strains under intracellular nitrogen limitation by S. Anastassiadis; A. Aivasidis; C. Wandrey (pp. 81-87).
A suitable strain and important factors influencing citric acid formation in yeasts were identified. Candida oleophila ATCC 20177 was chosen as the best citric acid producer from several Candida strains. Yields of 50 g/l citric acid were produced in shake flask and 80 g/l in fed-batch fermentations with 1.5 and 3 g/l NH4Cl under non-optimized conditions. Ammonium nitrogen was identified as the limiting substrate for citrate formation. Citric acid excretion begins a few hours after exhaustion of nitrogen in the medium. The importance of intracellular nitrogen limitation was clarified by elemental analysis of C. oleophila biomass. The nitrogen content of C. oleophila biomass decreased from 7.45% during the growth phase to 3.96% in the production phase. The biomass contained less carbon and more trace elements in the growth phase compared with the production phase. Relatively high intracellular NH4 + concentration of about 1.2 mg/g biomass (~37.4 mM) was found during the production phase. The low intracellular nitrogen content and increase of intracellular NH4 + concentration, possibly caused by proteolysis following extracellular nitrogen exhaustion, trigger citric acid production. Intracellular nitrogen limitation and the increase in intracellular NH4 + concentration are the most important factors influencing citric acid formation in yeasts.
Screening of facultative anaerobic bacteria utilizing D-xylose for xylitol production by S. Rangaswamy; F. Agblevor (pp. 88-93).
Seventeen cultures belonging to three genera of facultative bacteria (Serratia, Cellulomonas, and Corynebacterium) were screened for the production of xylitol, a sugar alcohol used as a sweetener in the pharmaceutical and food industries. The bacterial strains that utilized D-xylose for growth were investigated for xylitol production. A chromogenic assay of both solid and liquid cultures showed that ten of the 17 bacteria screened could grow on D-xylose and produce detectable quantities of xylitol during 24–96 h of fermentation. Among the screened cultures, Corynebacterium sp. B-4247 produced the highest amount of xylitol. In addition, the ten bacterial cultures that initially produced xylitol were studied for the effect of the environmental factors, such as temperature, concentration of D-xylose and aeration, on xylitol production.
Enzymatic (R)-phenylacetylcarbinol production in benzaldehyde emulsions by B. Rosche; N. Leksawasdi; V. Sandford; M. Breuer; B. Hauer; P. Rogers (pp. 94-100).
(R)-Phenylacetylcarbinol [(R)-PAC)] is the chiral precursor for the production of the pharmaceuticals ephedrine and pseudoephedrine. Reaction conditions were improved to achieve increased (R)-PAC levels in a simple batch biotransformation of benzaldehyde emulsions and pyruvate, using partially purified pyruvate decarboxylase (PDC) from the filamentous fungus Rhizopus javanicus NRRL 13161 as the catalyst. Lowering the temperature from 23°C to 6°C decreased initial rates but increased final (R)-PAC concentrations. Addition of ethanol, which increases benzaldehyde solubility, was not beneficial for (R)-PAC production. It was established that proton uptake during biotransformation increases the pH above 7 thereby limiting (R)-PAC production. For small-scale studies, biotransformations were buffered with 2–2.5 M MOPS (initial pH 6.5). High concentrations of MOPS as well as some alcohols and KCl stabilised PDC. A balance between PDC and substrate concentrations was determined with regards to (R)-PAC production and yields on enzyme and substrates. R. javanicus PDC (7.4 U/ml) produced 50.6 g/l (337 mM) (R)-PAC in 29 h at 6°C with initial 400 mM benzaldehyde and 600 mM pyruvate. Molar yields on consumed benzaldehyde and pyruvate were 97% and 59%, respectively, with 17% pyruvate degraded and 24% converted into acetaldehyde and acetoin; 43% PDC activity remained, indicating reasonable enzyme stability at high substrate and product concentrations.
Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy by Y. Li; J. Hugenholtz; J. Chen; S.-Y. Lun (pp. 101-106).
The effect of agitation speeds on the performance of producing pyruvate by a multi-vitamin auxotrophic yeast, Torulopsis glabrata, was investigated in batch fermentation. High pyruvate yield on glucose (0.797 g g–1) was achieved under high agitation speed (700 rpm), but the glucose consumption rate was rather low (1.14 g l–1 h–1). Glucose consumption was enhanced under low agitation speed (500 rpm), but the pyruvate yield on glucose decreased to 0.483 g g–1. Glycerol production was observed under low agitation speed and decreased with increasing agitation speed. Based on process analysis and carbon flux distribution calculation, a two-stage oxygen supply control strategy was proposed, in which the agitation speed was controlled at 700 rpm in the first 16 h and then switched to 500 rpm. This was experimentally proven to be successful. Relatively high concentration of pyruvate (69.4 g l–1), high pyruvate yield on glucose (0.636 g g–1), and high glucose consumption rate (1.95 g l–1 h–1) were achieved by applying this strategy. The productivity (1.24 g l–1 h–1) was improved by 36%, 23% and 31%, respectively, compared with fermentations in which agitation speeds were kept constant at 700 rpm, 600 rpm, and 500 rpm. Experimental results indicate that the difference between the performances for producing pyruvate under a favorable state of oxygen supply (dissolved oxygen concentration >50%) was caused by the different regeneration pathways of NADH generated from glycolysis.
Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy
by Y. Li; J. Hugenholtz; J. Chen; S.-Y. Lun (pp. 107-107).
Detection of two distinct substrate-dependent catabolic responses in yeast cells using a mediated electrochemical method by K. Baronian; A. Downard; R. Lowen; N. Pasco (pp. 108-113).
Mediated electrochemical detection of catabolism in prokaryotic cells is well documented; however, the application of this technique to eukaryotic cells has received less attention. Two catabolic substrate-dependent mediated electrochemical signals were detected in the yeast Saccharomyces cerevisiae. The signal using a single hydrophilic mediator (ferricyanide) is small whereas the response using a double mediator system comprising a hydrophilic and a lipophilic mediator (ferricyanide and menadione) is up to three orders of magnitude larger. The behaviour of each response during cell ageing is different: the single mediator response increases whereas the double mediator response decreases. This difference indicates that the two signals originate at different points in the catabolic pathways. In S. cerevisiae the double mediator response is proposed to originate from the reduction of the lipophilic mediator by NADPH produced in the pentose phosphate pathway. The single mediator signal arises from reduction of the hydrophilic mediator by an extracellular redox species produced in response to the presence of glucose.
Flocculent activity of a recombinant protein from Moringa oleifera Lam. seeds by M. Broin; C. Santaella; S. Cuine; K. Kokou; G. Peltier; T. Joët (pp. 114-119).
Seeds of the tropical tree Moringa oleifera contain small storage proteins able to flocculate particles in suspension in water. The cDNA encoding one of these flocculent proteins, MO2.1, was cloned and the recombinant protein was expressed in Escherichia coli. The flocculent activity of the purified recombinant MO2.1 was assayed on clays and bacteria using light and confocal microscopy and GFP-overexpressing bacteria. We show that MO2.1 is able to aggregate montmorillonite clay particles as well as gram-positive and gram-negative bacteria. We discuss the use of recombinant proteins to study flocculating properties and improve water purification processes.
Hydrolysis of steryl esters by a lipase (Lip 3) from Candida rugosa by M. Tenkanen; H. Kontkanen; R. Isoniemi; P. Spetz; B. Holmbom (pp. 120-127).
A well-known lipase, Lip 3 of Candida rugosa, was purified to homogeneity from a commercial lipase preparation, using hydrophobic interaction and anion exchange chromatography. Lip 3, which has been reported to act on cholesteryl esters, was also found to be active on plant-derived steryl esters. Lip 3 had optimal activity at pH 5–7 and below 55 °C. It was able to hydrolyse steryl esters totally in a clear micellar aqueous solution. However, the action on a dispersed colloidal steryl ester solution was limited and only about half of the steryl esters were degraded. The degree of hydrolysis was not improved by addition of fresh enzyme. The composition of released fatty acids and sterols was, however, almost identical to that obtained by alkaline hydrolysis, showing that all the different steryl esters were hydrolysed equally and that none of the individual components were responsible for incomplete hydrolysis. Thus, it appeared that the physical state of the colloidal steryl ester dispersion limited the action of Lip 3. Wood resins contain both triglycerides and steryl esters among the hydrophobic components, which create problems in papermaking. The simultaneous enzymatic hydrolysis of triglycerides and steryl ester is therefore of considerable interest and Lip 3 is the first enzyme reported to act on both triglycerides and steryl esters.
Purification and characterization of the monooxygenase catalyzing sulfur-atom specific oxidation of dibenzothiophene and benzothiophene from the thermophilic bacterium Paenibacillus sp. strain A11–2 by J. Konishi; Y. Ishii; T. Onaka; K. Maruhashi (pp. 128-133).
A benzothiophene (BT) and dibenzothiophene (DBT) monooxygenase (TdsC), which catalyzes the oxidation of the sulfur atoms in BT and DBT molecules, was purified from Paenibacillus sp. strain A11–2. The molecular mass of the purified enzyme and its subunit were determined to be 200 kDa and 43 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, indicating a tetrameric structure. The N-terminal amino acid sequence of the purified TdsC completely matched the amino acid sequence deduced from the nucleotide sequence of the tdsC gene reported previously [Ishii et al. (2000) Biophys Biochem Res Commun 270:81–88]. The optimal temperature and pH for the TdsC reaction were 65°C and pH 9, respectively. TdsC required NADH, FMN and TdsD, a NADH-dependent FMN oxidoreductase, for its activity, as was observed for TdsA. FAD, lumiflavin and/or NADPH had some effect on the maintenance of TdsC activity. A comparison of the substrate specificity of TdsC and DszC, the homologous monooxygenase purified from Rhodococcus erythropolis strain KA2–5–1, demonstrated a contrasting pattern towards alkylated DBTs and BTs.
The effect of a disrupted yhjQ gene on cellular morphology and cell growth in Escherichia coli by M. Kim; S. Park; S. Cho; W. Lim; S. Ryu; C. An; S. Hong; Y. Park; G. Kahng; J. Kim; H. Kim; H. Yun (pp. 134-138).
The 5′ upstream region of the cellulose synthase operon (bcs operon) has been isolated by cloning from Escherichia coli. A gene encoding YhjQ is located 1.0 kb upstream of the bcs operon in E. coli. The function of YhjQ remains unknown. Insertional inactivation of the yhjQ gene causes abnormal cell division, resulting in incomplete partition of the chromosome and filamentous cells of various sizes. These results suggest that the product of yhjQ may affect normal doubling and cellular morphology.
MoeA, an enzyme in the molybdopterin synthesis pathway, is required for rifamycin SV production in Amycolatopsis mediterranei U32 by W. Wang; W. Zhang; J. Lu; Y. Yang; J. Chiao; G. Zhao; W. Jiang (pp. 139-146).
Rifamycin SV contains one amide nitrogen atom at its C7N moiety. Earlier labeling studies suggested that nitrogen might be incorporated from a pathway involved in a molybdenum-dependent nitrate reductase. However, no genetic evidence is available thus far. The structural gene moeA, which is involved in molybdopterin synthesis in various organisms, has been cloned from rifamycin SV-producing Amycolatopsis mediterranei strain U32. The amino acid sequence deduced from the moeA gene showed significant similarity to members of the MoeA protein family and contains all the structural features that are highly conserved in the putative functional domains of MoeA proteins. Southern hybridization showed that there is only one moeA gene in the A. mediterranei genome. To further investigate the possible physiological function of the moeA gene, a double crossover gene replacement was achieved by inserting an aparmycin resistance gene into moeA in the A. mediterranei U32 chromosome. Phenotype analysis showed that the moeA gene is required for A. mediterranei growth in a minimal medium with nitrate as sole nitrogen source, possibly because nitrate reductase activity is diminished due to disruption of the moeA gene. Compared to the wild type strain, moeA-disrupted mutants lost 95% of their rifamycin SV production capacity in complex fermentation media. The results demonstrate that the moeA gene is necessary for rifamycin SV production in A. mediterranei, and that the nitrogen assimilation pathway involved in nitrate reductase is the major pathway for the genesis of the amide nitrogen atom in the rifamycin SV molecule.
Reductive effect of H2 uptake and poly-β-hydroxybutyrate formation on nitrogenase-mediated H2 accumulation of Rhodobacter sphaeroides according to light intensity by I.-H. Lee; J. Park; D. Kho; M.-S. Kim; J. Lee (pp. 147-153).
Nitrogenase-mediated H2 accumulation of Rhodobacter sphaeroides under photoheterotrophic conditions is reduced directly by the hydrogenase activity catalyzing H2 uptake and indirectly by energy-demanding metabolic processes such as poly-β-hydroxybutyrate (PHB) formation. H2 accumulation of R. sphaeroides was examined during cell growth under illumination of 15, 7, and 3 W/m2. Mutations in either hupSL (H2-uptake hydrogenase) or phbC (PHB synthase) had no effect on nitrogenase activity. The nitrogenase activity of R. sphaeroides grown at 15 W/m2, however, was 70% higher than that of cells grown at 3 W/m2, while the H2-uptake hydrogenase activity was approximately 3-fold higher in the same comparison. Accordingly, H2 uptake by hydrogenase, monitored by measuring the difference in H2 accumulation between a hupSL-deletion mutant and the corresponding parental strain, appeared to reach a maximum level as illumination was increased to 15 W/m2. On the other hand, the surplus energy due to lack of PHB formation led to a fixed increase in H2 accumulation independent of light intensity, reflecting the fact that the cellular PHB content was not changed significantly depending on light intensity. Therefore, H2 uptake by hydrogenase should be suppressed to achieve higher H2 accumulation of R. sphaeroides, especially at 15 W/m2.
Poly(ethylene glycol)-mediated molar mass control of short-chain- and medium-chain-length poly(hydroxyalkanoates) from Pseudomonas oleovorans by R. Ashby; D. Solaiman; T. Foglia (pp. 154-159).
Three strains of Pseudomonas oleovorans, a well known poly(hydroxyalkanoate) (PHA) producer, were tested for the ability to control PHA molar mass and end group structure by addition of poly(ethylene glycol) (PEG) to the fermentation medium. Each strain of P. oleovorans – NRRL B-14682 (B-14682), NRRL B-14683 (B-14683), and NRRL B-778 (B–778) – synthesized a different type of PHA from oleic acid when cultured under identical growth conditions. Strain B-14682 produced poly(3-hydroxybutyrate) (PHB), while B-14683 synthesized a medium-chain-length PHA (mcl-PHA) with a repeat unit composition ranging from C4 to C14 and some mono-unsaturation in the C14 alkyl side chains. Strain B-778 synthesized a mixture of PHB (95 mol%) and mcl-PHA (5 mol%). The addition of 0.5% (v/v) PEG (Mn =200 g/mol, PEG-200) to the fermentation broth of strains B-14682 and B-778 resulted in chain termination through esterification at the carboxyl terminus of the PHB with PEG chain segments, thus reducing the molar mass by 54% and 23%, respectively. The molar mass of the mcl-PHA produced by strains B-14683 and B-778 also showed a 34% and 47% reduction in the presence of PEG-200, respectively, but no evidence of esterification was present. PEG-400 (Mn =400 g/mol) had a reduced effect on PHA molar mass. In fact, the molar masses of the mcl-PHA derived from strain B-14683 and both the PHB and mcl-PHA from B-778 were unchanged by PEG-400. In contrast, the PHB produced by B-14682 showed a 35% reduction in molar mass in the presence of PEG-400.
Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1 by K. Tanaka; A. Komiyama; K. Sonomoto; A. Ishizaki; S. Hall; P. Stanbury (pp. 160-167).
In lactic acid bacteria, pentoses are metabolized via the phosphoketolase pathway, which catalyzes the cleavage of D-xylulose-5-phosphate to equimolar amounts of glyceraldehyde 3-phosphate and acetylphosphate. Hence the yield coefficient of lactate from pentose does not exceed 1.0 mol/mol, while that of Lactococcus lactis IO-1(JCM7638) at high D-xylose concentrations often exceeds the theoretical value. This suggests that, in addition to the phosphoketolase pathway, L. lactis IO-1 may possess another metabolic pathway that produces only lactic acid from xylose. In the present study, the metabolism of xylose in L. lactis IO-1 was deduced from the product formation and enzyme activities of L. lactis IO-1 in batch culture and continuous culture. During cultivation with xylose concentrations above ca. 50 g/l, the yield coefficient of L-lactate exceeded 1.0 mol/mol while those of acetate, formate and ethanol were very low. At xylose concentrations less than 5 g/l, acetate, formate and ethanol were produced with yield coefficients of about 1.0 mol/mol, while L-lactate was scarcely produced. In cells grown at high xylose concentrations, a marked decrease in the specific activities of phosphoketolase and pyruvate formate lyase (PFL), and an increase in those of transketolase and transaldolase were observed. These results indicate that in L. lactis IO-1 xylose may be catabolized by two different pathways, the phosphoketolase pathway yielding acetate, formate and ethanol, and the pentose phosphate (PP)/glycolytic pathway which converts xylose to L-lactate only. Furthermore, it was deduced that the change in the xylose concentration in the culture medium shifts xylulose 5-phosphate metabolism between the phosphoketolase pathway and the PP/glycolytic pathway in L. lactis IO-1, and pyruvate metabolism between cleavage to acetyl-CoA and formic acid by PFL and the reduction to L-lactate by lactate dehydrogenase.
Bifidobacterium longum ATCC 15707 cell production during free- and immobilized-cell cultures in MRS-whey permeate medium by Y. Doleyres; C. Paquin; M. LeRoy; C. Lacroix (pp. 168-173).
Bifidobacterium longum ATCC 15707 cell production was studied in MRS medium supplemented with whey permeate (MRS-WP) during free-cell batch fermentations and continuous immobilized-cell cultures. Very high populations were measured after 12 h batch cultures in MRS-WP medium controlled at pH 5.5 (1.7±0.5×1010 cfu/ml), approximately 2-fold higher than in non-supplemented MRS. Our study showed that WP is a low-cost source of lactose and other components that can be used to increase bifidobacteria cell production in MRS medium. Continuous fermentation in MRS-WP of B. longum immobilized in gellan gum gel beads produced the highest cell concentrations in the effluent (4.9±0.9×109 cfu/ml) at a dilution rate (D) of 0.5 h–1. However, maximal volumetric productivity (6.9±0.4×109 cfu ml–1h–1) during continuous cultures was obtained at D =2.0 h–1, and was approximately 9.5-fold higher than during free-cell batch cultures at an optimal pH of 5.5 (7.2×108 cfu ml–1h–1).
Variability in nitrogen regulation of aflatoxin production by Aspergillus flavus strains by K. Ehrlich; P. Cotty (pp. 174-178).
Aflatoxins are toxic and carcinogenic metabolites of several Aspergillus species. The effect of nitrate on aflatoxin production and expression of the key regulatory genes involved in aflatoxin biosynthesis, aflR and aflJ, were compared among isolates of the SB and SBG strains of A. flavus. Aflatoxin production by two of the three strain SB isolates did not differ significantly between the two media tested, whereas for SBG A. flavus isolates, the level of aflatoxins in buffered nitrate medium was as much as 20-fold lower than in ammonium salts medium. Expression of aflR was not significantly affected by growth of cultures in nitrate medium for most of the isolates. However, on nitrate medium, expression of aflJ was 2.6-fold higher for the SB isolates than it was on ammonium salts medium, whereas for the SBG isolates aflJ expression was 2-fold lower on nitrate than on ammonium salts medium. This difference may result from the presence in the aflJ/aflR intergenic region of SBG isolates of fewer putative binding sites (HGATAR sites) for AreA, the positive-acting, wide domain transcription factor involved in regulation of nitrogen metabolism.
Cis/trans isomerisation of unsaturated fatty acids in a cardiolipin synthase knock-out mutant of Pseudomonas putida P8 by A. von Wallbrunn; H. Heipieper; F. Meinhardt (pp. 179-185).
The gene encoding cardiolipin synthase (cls) from the phenol-degrading bacterium Pseudomonas putida P8, which rapidly adapts its membrane lipids to the presence of organic solvents by cis/trans isomerisation of unsaturated fatty acids, was isolated and completely sequenced. The functionality of the predicted gene product was proven by constructing a knock-out mutant that was significantly reduced in its growth rate both at elevated temperatures and in the presence of membrane-active solvents. Though the mutant showed a clear phenotype it was still able to synthesise trace amounts of cardiolipin. As an increase in cardiolipin (diphosphatidylglycerol) content is known to function as a long term membrane adaptation mechanism in pseudomonads, we tested whether the mutant compensates for the lack of the Cls by increased cis/trans isomerisation of unsaturated fatty acids. Increase in cis/trans isomerisation of unsaturated fatty acids was observed for the mutant at zero and low concentrations of 4-chlorophenol; however, cis/trans isomerisation is not able to fully compensate for the lack of cardiolipin production. Possibly, other long-term adaptation mechanisms are instrumental in compensating for the missing cardiolipin synthesis. As the cis/trans isomerase is activated similarly in the mutant and the wildtype, cis/trans isomerisation and cardiolipin production do not display mutual dependency.
Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae by W.-J. Lee; M.-D. Kim; Y.-W. Ryu; L. Bisson; J.-H. Seo (pp. 186-191).
Zero trans-influx assays of glucose and xylose were performed using Saccharomyces cerevisiae to investigate transport characteristics under high and low glucose conditions. Under high glucose conditions, most glucose was transported by the low-affinity transporter. The high-affinity transporter was expressed under low glucose conditions, transporting over 50% glucose. Inhibition kinetics revealed that xylose was transported by both high- and low-affinity glucose transporters. Affinities of both glucose transporters for xylose were very low under high glucose condition but increased to a similar level to glucose under low glucose condition. The maximum rate of xylose transport increased by 85%, while an overall maximum glucose transport rate decreased by 42% under low glucose condition, indicating the presence of other transport system for sugars except for glucose. It was suggested that expression of the high-affinity transporter and increased affinity of glucose transporters for xylose under low glucose condition would provide a fermentation strategy for enhancing the productivity of xylitol by recombinant S. cerevisiae harboring the xylose reductase gene.
Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates by R. Sani; B. Peyton; W. Smith; W. Apel; J. Petersen (pp. 192-199).
The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp. (WS01, WS18, and ES5) and a known Cellulomonas strain (Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions. In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI). In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) [Fe(III)-NTA] from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM. All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls. This is the first report of Cellulomonas sp. reducing Fe(III) and U(VI). Further, this is the first report of Cellulomonas spp. coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI).
Oxidation of dibenzo-p-dioxin, dibenzofuran, biphenyl, and diphenyl ether by the white-rot fungus Phlebia lindtneri by T. Mori; R. Kondo (pp. 200-205).
Hydroxylation of dibenzo-p-dioxin (DD), dibenzofuran (DF), biphenyl (BP) and diphenyl ether (DPE) by the white-rot fungus Phlebia lindtneri GB-1027 was studied. DD and DF were rapidly degraded in a culture of P. lindtneri. The initial oxidation products were identified by gas chromatography-mass spectrometry. P. lindtneri oxidized DD to 2-hydroxy-DD, and DF to 2- and 3-hydroxy-DF. BP and DPE were also oxidized to p-hydroxy-BP and p-hydroxy-DPE, respectively. The oxidation catalyzed by P. lindtneri with each substrate was position-specific, because the hydroxyl group was introduced to the molecular edge of every substrate. Significant inhibition of the degradation of DD and DF was observed in incubation with the cytochrome P-450 monooxygenase inhibitors 1-aminobenzotriazole and piperonyl butoxide. These experiments with cytochrome P-450 inhibitors, and formation of the mono-hydroxyl metabolites suggest that P. lindtneri initially oxidizes DD, DF, BP, and DPE by a cytochrome P-450 monooxygenase and that it directly introduces a hydroxyl group to each of these substrates.
Monitoring of a pyrite-oxidising bacterial population using DNA single-strand conformation polymorphism and microscopic techniques by F. Battaglia-Brunet; M. Clarens; P. d'Hugues; J. Godon; S. Foucher; D. Morin (pp. 206-211).
The single-strand conformation polymorphism (SSCP) technique was used to study the evolution of a bacterial consortium during the batch oxidation of a cobaltiferous pyrite in two types of bio-reactor: a bubble column and a classical stirred tank. Sequencing 16S rDNA revealed the presence of three organisms affiliated to Leptospirillum ferrooxidans, Acidithiobacillus thiooxidans and Sulfobacillus thermosulfidooxidans, respectively. Attempts were made to determine the proportions of bacteria attached to solid particles or freely suspended in the medium using a combination of PCR-SSCP and a microscopic technique. Ac. thiooxidans-related bacteria were dominant in the liquid during the early phase of the batch, but were later supplanted by L. ferrooxidans-related bacteria. L. ferrooxidans-related organisms were always in the majority on the solids. The growth of S. thermosulfidooxidans-related bacteria seemed to be favoured by the bubble-column reactor.
Removal and mineralization of polycyclic aromatic hydrocarbons by litter-decomposing basidiomycetous fungi by K. Steffen; A. Hatakka; M. Hofrichter (pp. 212-217).
Nine strains of litter-decomposing fungi, representing eight species of agaric basidiomycetes, were tested for their ability to remove a mixture of three polycyclic aromatic hydrocarbons (PAHs) (total 60 mg l–1) comprising anthracene, pyrene and benzo(a)pyrene (BaP) in liquid culture. All strains were able to convert this mixture to some extent, but considerable differences in degradative activity were observed depending on the species, the Mn(II) concentration, and the particular PAH. Stropharia rugosoannulata was the most efficient degrader, removing or transforming BaP almost completely and about 95% of anthracene and 85% of pyrene, in cultures supplemented with 200 µM Mn(II), within 6 weeks. In contrast less than 40, 18, and 50% BaP, anthracene and pyrene, respectively, were degraded in the absence of supplemental Mn(II). In the case of Stropharia coronilla, the presence of Mn(II) led to a 20-fold increase of anthracene conversion. The effect of manganese could be attributed to the stimulation of manganese peroxidase (MnP). The maximum activity of MnP increased in S. rugosoannulata cultures from 10 U l–1 in the absence of Mn(II) to 320 U l–1 in Mn(II)-supplemented cultures. The latter degraded about 6% of a 14C-labeled BaP into 14CO2 whereas only 0.7% was mineralized in the absence of Mn(II). In solid-state straw cultures, S. rugosoannulata, S. coronilla and Agrocybe praecox mineralized between 4 and 6% of 14C-labeled BaP within 12 weeks.
Evaluation of support materials for the surface immobilization and decoloration of amaranth by Trametes versicolor by M. Shin; T. Nguyen; J. Ramsay (pp. 218-223).
The ability of Trametes versicolor ATCC 20869 to colonize several natural and synthetic materials (wheat straw, jute, hemp, maple woodchips, and nylon and polyethylene teraphthalate fibers) and to subsequently decolorize amaranth was evaluated. Jute was found to be the best support material as T. versicolor grew well on it without color leaching from the support and without loss of the jute's integrity over a 4 week period. The fungus immobilized on jute, straw and hemp decolorized amaranth (50 mg l–1) at a rate of about 5 mg l–1 h–1 without glucose being added. When 1 g l–1 glucose was added, the dye was degraded more quickly (about 8 mg l–1 h–1). Decoloration did not occur in a suspension culture without glucose. As the number of decoloration cycles increased, the rate of decoloration decreased. This rate was restored to its original level after the biomass was incubated in fresh growth medium for 5 days. With all immobilization supports, the toxicity of the medium before and after decoloration was the same or lower.
Microbial community changes during organic solid waste treatment analyzed by double gradient-denaturing gradient gel electrophoresis and fluorescence in situ hybridization by S. Haruta; M. Kondo; K. Nakamura; H. Aiba; S. Ueno; M. Ishii; Y. Igarashi (pp. 224-231).
The bacterial community present during semicontinuous treatment of organic solid waste under alkaline and high-temperature conditions was studied. PCR-amplified 16S rDNA fragments were analyzed by double gradient-denaturing gradient gel electrophoresis (DGGE). The band pattern was stable during the steady state of the treatment phase, and the major bands resulting from individual treatments had the same DNA sequence with good reproducibility. No sequence in the DNA database of isolated bacteria showed close similarity to this sequence, the closest relative being Bacillus licheniformis with less than 97% similarity. The conditions for fluorescence in situ hybridization (FISH) were determined without the need to obtain extracts of the bacterial cells. An oligonucleotide probe was designed to detect the microorganisms found in the DGGE analysis. FISH analysis showed that the bacterium corresponding to the major bands accounted for 30% of the total eubacterial cell count at the steady state. These results indicate that this bacterium is a key microorganism in the biodegradation process.