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Applied Microbiology and Biotechnology (v.57, #3)

Unusual enzymes involved in five pathways of glutamate fermentation by W. Buckel (pp. 263-273).

Anaerobic bacteria from the orders Clostridiales and Fusobacteriales are able to ferment glutamate by at least five different pathways, most of which contain enzymes with radicals in their catalytic pathways. The first two pathways proceed to ammonia, acetate and pyruvate via the coenzyme B₁₂-dependent glutamate mutase, which catalyses the re-arrangement of the linear carbon skeleton to that of the branched-chain amino acid (2S,3S)-3-methylaspartate. Pyruvate then disproportionates either to CO₂ and butyrate or to CO₂, acetate and propionate. In the third pathway, glutamate again is converted to ammonia, CO₂, acetate and butyrate. The key intermediate is (R)-2-hydroxyglutaryl-CoA, which is dehydrated to glutaconyl-CoA, followed by decarboxylation to crotonyl-CoA. The unusual dehydratase, containing an iron–sulfur cluster, is activated by an ATP-dependent one-electron reduction. The remaining two pathways require more then one organism for the complete catabolism of glutamate to short chain fatty acids. Decarboxylation of glutamate leads to 4-aminobutyrate, which is fermented by a second organism via the fourth pathway to acetate and butyrate, again mediated by an unusual dehydratase which catalyses the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The fifth pathway is the only one without decarboxylation, since the γ-carboxylate of glutamate is reduced to the amino group of δ-aminovalerate, which then is fermented to acetate, propionate and valerate. The pathway involves the oxidative dehydration of 5-hydroxyvaleryl-CoA to 2,4-pentadienoyl-CoA followed by reduction to 3-pentenoyl-CoA and isomerisation to 2-pentenoyl-CoA.

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs by M. Sisido; T. Hohsaka (pp. 274-281).

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.

Antimicrobial properties of Allium sativum (garlic) by J. C. Harris; S. Cottrell; S. Plummer; D. Lloyd (pp. 282-286).

Although garlic has been used for its medicinal properties for thousands of years, investigations into its mode of action are relatively recent. Garlic has a wide spectrum of actions; not only is it antibacterial, antiviral, antifungal and antiprotozoal, but it also has beneficial effects on the cardiovascular and immune systems. Resurgence in the use of natural herbal alternatives has brought the use of medicinal plants to the forefront of pharmacological investigations, and many new drugs are being discovered. This review aims to address the historical use of garlic and its sulfur chemistry, and to provide a basis for further research into its antimicrobial properties.

Photobioreactors: production systems for phototrophic microorganisms by O. Pulz (pp. 287-293).

Microalgae have a large biotechnological potential for producing valuable substances for the feed, food, cosmetics and pharmacy industries as well as for biotechnological processes. The design of the technical and technological basis for photobioreactors is the most important issue for economic success in the field of phototrophic biotechnology. For future applications, open pond systems for large-scale production seem to have a lower innovative potential than closed systems. For high-value products in particular, closed systems of photobioreactors seem to be the more promising field for technical developments despite very different approaches in design.

Perspectives in the biological function, the technical and therapeutic application of bone morphogenetic proteins by A. Hoffmann; H. Weich; G. Gross; G. Hillmann (pp. 294-308).

The bone morphogenetic proteins (BMPs) belong to the transforming growth factor β superfamily of growth and differentiation factors and have been characterized by their ability to induce new bone formation in ectopic (non-skeletal) sites. BMPs are secreted molecules and are key regulators in early embryogenesis and organogenesis. One of the many functions of BMPs is to induce cartilage, bone, and connective tissue formation in vertebrates. This osteo-inductive capacity of BMPs has long been considered very promising for applications in bone repair, in the treatment of skeletal diseases, and in oral applications such as dentiogenesis and cementogenesis during regeneration of periodontal wounds. We discuss here biological roles of the BMPs in the organism and their signaling cascades leading to bone and cartilage formation in particular. It is also the aim of this review to evaluate the potential and the problems of BMPs in skeletal tissue engineering for the regeneration of bone damaged by disease or trauma and to serve as therapeutic agents for periodontal defects.

Biotransformation of benzaldehyde into (R)-phenylacetylcarbinol by filamentous fungi or their extracts by B. Rosche; V. Sandford; M. Breuer; B. Hauer; P. Rogers (pp. 309-315).

Extracts of 14 filamentous fungi were examined regarding their potential for production of (R)-phenylacetylcarbinol [(R)-PAC], which is the chiral precursor in the manufacture of the pharmaceuticals ephedrine and pseudoephedrine. Benzaldehyde and pyruvate were transformed at a scale of 1.2 ml into PAC by cell-free extracts of all selected strains, covering the broad taxonomic spectrum of Ascomycota, Zygomycota and Basidiomycota. Highest final PAC concentrations were obtained with the extracts of Rhizopus javanicus and Fusarium sp. [78–84 mM (11.7–12.6 g/l) PAC within 20 h from initial substrate concentrations of 100 mM benzaldehyde and 150 mM pyruvate]. (R)-PAC was in about 90–93% enantiomeric excess. Rhizopus javanicus had the advantage of faster growth than Fusarium sp. Rhizopus javanicus mycelia were used as an example in a biotransformation process based on whole cells and benzaldehyde and glucose as substrates. The substrate pyruvate was generated through the fungal fermentation of glucose. Only 19 mM PAC (2.9 g/l) were produced within 8 h from 80 mM benzaldehyde, with evidence of significant benzyl alcohol production.

A perfusion–cell bleeding culture strategy for enhancing the productivity of eicosapentaenoic acid by Nitzschia laevis by Z.-Y. Wen; F. Chen (pp. 316-322).

A perfusion–cell bleeding culture strategy was developed for enhancing the productivity of eicosapentaenoic acid (EPA) by the diatom Nitzschia laevis. As the strategy combined the concepts of continuous culture and perfusion culture, it allowed continuously and simultaneously harvesting the algal cells and removing inhibitory compounds during the cultivation. Compared with a single operation of continuous culture, the perfusion–cell bleeding culture greatly enhanced the steady-state biomass concentration, biomass productivity, EPA yield, EPA productivity and glucose utilization efficiency. The perfusion–cell bleeding culture also allowed higher biomass productivity and EPA productivity than the single perfusion culture did. At a bleeding rate of 0.67 day^–1 and a perfusion rate of 0.6 day^–1, the EPA productivity achieved 175 mg l^–1day^–1, which is the highest ever reported in microalgal cultures.

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures to ethanol and sorbitol by C. Shene; S. Bravo (pp. 323-328).

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures were carried out at different operational conditions (aeration, feed rate and substrate concentration) to test their effects on the system productivity. In these fermentations, the main products were ethanol and sorbitol. Kinetic parameters were calculated using the experimental data. However, parameters in the sorbitol synthesis rate were estimated from data recorded in different experiments in order to avoid the effect of the simultaneous cell growth and ethanol synthesis. In this case, the crude cell extract was used as source of the enzyme responsible for the sorbitol synthesis. The highest degree of conversion of fructose into sorbitol obtained with the extract was equal to 71% in a sugar mixture with an initial concentration of 200 g/l. Results obtained in the fed-batch fermentations showed that aeration of the culture has a positive effect on the final biomass concentration. However, final ethanol concentration is lower under aerated conditions. The best sugar yields to biomass and ethanol were 0.032 and 0.411 g/g, respectively. On the other hand, the highest sorbitol yield in the fed-batch fermentations was 0.148 g/g.

Highly efficient Aerococcus viridans L-α-glycerophosphate oxidase production in the presence of H₂O₂-decomposing agent: purification and kinetic characterization by S. Streitenberger; J. López-Mas; A. Sánchez-Ferrer; F. García-Carmona (pp. 329-333).

Glycerophosphate oxidase was purified from Aerococcus viridans cells by phase partitioning in Triton X-114, ammonium sulfate fractionation, FPLC ion-exchange chromatography and FPLC hydrophobic-interaction chromatography. The purification achieved from a crude extract of A. viridans was 38-fold with a 32% recovery of activity. Under the growth conditions used, A. viridans strain CECT 978 proved to be an excellent glycerophosphate-oxidase producer, with enzyme production 2,800-fold greater than that described in the literature for the same microorganism. The culture medium used in the present work is that commonly used for cultivation of this microorganism, except that an H₂O₂-decomposing enzyme was added. The addition of catalase to the growth medium had a clear effect on the growth rate. Furthermore, methylglyoxal, a metabolite that is formed enzymatically from triose phosphates, was found to be an inactivator of glycerophosphate oxidase activity.

Shrimp chitin as substrate for fungal chitin deacetylase by N. Win; W. Stevens (pp. 334-341).

The fungal chitin deacetylases (CDA) studied so far are able to perform heterogeneous enzymatic deacetylation on their solid substrate, but only to a limited extent. Kinetic data show that about 5–10% of the N-acetyl glucosamine residues are deacetylated rapidly. Thereafter enzymatic deacetylation is slow. In this study, chitin was exposed to various physical and chemical conditions such as heating, sonicating, grinding, derivatization and interaction with saccharides and presented as a substrate to the CDA of the fungus Absidia coerulea. None of these treatments of the substrate resulted in a more efficient enzymatic deacetylation. Dissolution of chitin in specific solvents followed by fast precipitation by changing the composition of the solvent was not successful either in making microparticles that would be more accessible to the enzyme. However, by treating chitin in this way, a decrystallized chitin with a very small particle size called superfine (SF) chitin could be obtained. This SF chitin, pretreated with 18% formic acid, appeared to be a good substrate for fungal deacetylase. This was confirmed both by enzyme-dependent deacetylation measured by acetate production as well as by isolation and assay for the degree of deacetylation (DD). In this way chitin (10% DD) was deacetylated by the enzyme into chitosan with DD of 90%. The formic acid treatment reduced the molecular weight of the polymeric chain from 2×10⁵ in chitin to 1.2×10⁴in the chitosan product. It is concluded that nearly complete enzymatic deacetylation has been demonstrated for low-molecular chitin.

Optimal design for the maximization of Penicillium cyclopium lipase production by G. Vanot; V. Deyris; M.-C. Guilhem; Phan R. Luu; L.-C. Comeau (pp. 342-345).

Penicillium cyclopium triacylglycerol lipase production was maximized in stationary batch culture. We used a surface response methodology based on a Doehlert experimental design to study the effect on the lipase activity released in the culture medium of the three most important factors: substrate concentration, pH and inoculum. Besides reducing the number of experiments required for optimization, this technique allowed us to quantify the lipase activity in any part of the experimental domain.We determined an optimal set of conditions for high lipase production: 1% substrate (corn steep), pH 5.5 and an inoculum of 10⁴ spores/ml. Between conditions giving the minimum and the maximum lipase production, we observed a nine-fold increase of both the predicted and measured values.

Influence of carbon source on α-amylase production by Aspergillus oryzae by M. Carlsen; J. Nielsen (pp. 346-349).

The influence of the carbon source on α-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of α-amylase, whereas addition of small amounts of glucose resulted in α-amylase production. A possible induction by α-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of α-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.

Cloning and characterization of the gene cahB encoding a cephalosporin C acetylhydrolase from Acremonium chrysogenum by J. Velasco; S. Gutiérrez; J. Casqueiro; F. Fierro; S. Campoy; J. Martín (pp. 350-356).

An important problem during the production of cephalosporin C by Acremonium chrysogenum is the hydrolysis of cephalosporin C to deacetylcephalosporin C, since the latter compound has no commercial value and represents an unwanted side-product. Characterization of the enzymatic process that gives rise to deacetylcephalosporin C will help to avoid the accumulation of this side-product. An extracellular cephalosporin C acetylhydrolase (CPC-AH) from Acremonium chrysogenum C10 was purified to near homogeneity. This enzyme had a molecular mass of 31 kDa, a pI of 4.0, and showed relatively little affinity for cephalosporin C (K _m 33.7 mM). We sequenced twenty amino acids at the amino-terminal end; a probe based on this sequence was then used to clone the cephalosporin acetylhydrolase (cahB) gene. cahB encodes a pre-protein of 383 amino acids with a deduced molecular mass of 38,228 Da. The sequenced 20 amino acids of the purified protein corresponded to amino acids 107–127 deduced from the cahB gene, suggesting that mature CPC-AH results from processing of the pre-protein after Gln-106. cahB is located on chromosome VIII of A. chrysogenum C10 and is not linked to the cephalosporin early or late gene clusters. It is expressed as a single 1.4-kb transcript after 72 h of cultivation. Expression declined in batch cultures after 120 h even though CPC-AH activity was observed until 144 h. The CPC-AH protein resembles other wide-spectrum substrate fungal esterases that are functionally related to serine proteases. The cahB gene does not seem to be related to the cephalosporin biosynthesis genes and encodes an esterase active on several substrates in addition to cephalosporin C.

Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7-aminodeacetoxycephalosporanic acid production. by J. Robin; M. Jakobsen; M. Beyer; H. Noorman; J. Nielsen (pp. 357-362).

The production of adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this process therefore represents a new route for the production of 7-ADCA, which serves as a precursor for the production of many semi-synthetic cephalosporins. In this study, one low- and one high-yielding strains were characterised and the specific productivities of ad-7-ADCA and by-products of the biosynthetic pathway were compared. The fluxes through the biosynthetic pathway were quantified and it was found that there was a 30% higher flux through the expandase in the high-yielding strain. In both strains, there was a significant degradation of adipate. Furthermore, the initial adipate concentration in batch cultures was shown to have a positive effect on the formation of ad-7-ADCA.

Replacement of arginine-171 and aspartate-453 in Streptomyces coelicolor malate synthase A by site-directed mutagenesis inactivates the enzyme by L.-L. Goh; P. Loke; T.-S. Sim (pp. 363-367).

Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA. Escherichia coli is known to possess two forms of malate synthase, A and G respectively. The recent elucidation of the E. coli malate synthase G crystal structure suggested two residues, Arg338 and Asp631, are essential for catalysis. Multiple sequence alignment of 26 known malate synthase enzymes revealed that the two proposed sites are highly conserved, despite the low homologies between the two distinct forms of the enzyme (13–18%). The conservation of these residues in both forms of malate synthase suggests that they possess a similar catalytic strategy. Thus, despite the absence of a three-dimensional structure for malate synthase A, the significance of this enzyme in the primary metabolic pathway has prompted the investigation of the involvement of the corresponding residues, Arg171 and Asp453, in Streptomyces coelicolor malate synthase A by site-directed mutagenesis. Heterologous expression in E. coli followed by purification of the constructed mutant proteins, Arg171Leu and Asp453Ala, were performed and subsequent enzyme assays of the purified mutant proteins indicated a significant loss of catalytic activity, thus attesting to the need for the corresponding conserved residues to maintain malate synthase functionality.

Activation of plasma membrane H⁺-ATPase by ammonium ions in Aspergillus niger by K. Jernejc; M. Legiša (pp. 368-373).

The addition of ammonium ions to Aspergillus niger cells originally growing on another nitrogen source resulted in rapid medium acidification. The addition of glucose or other fermentable sugars to the mycelium growing on glycerol did not have the same effect. The enzyme responsible for acidification seems to be plasma membrane H⁺-ATPase, which is most probably triggered by phosphorylation. Using specific activators and inhibitors, we tried to figure out which signalling pathway is involved in the process. No activation of H⁺-ATPase could be detected in the presence of diacylglycerol and other activators of protein kinase C, indicating that the stimulus is transmitted by another signalling chain. In the presence of inhibitors known to suppress the phosphatidyl–inositol signalling pathway, such as neomycin, compound 48/80 and calmidazolium, no increased H⁺-ATPase activity could be detected after the addition of ammonium ions. However, some tested inhibitors of the cAMP signalling pathway could not prevent activation of the enzyme by the stimulant. These results support the model in which ammonium-induced activation of proton extrusion in A. niger is mediated via the phosphatidyl–inositol signalling pathway, involving Ca²⁺/calmoduline-dependent protein kinase but not protein kinase C.

Effect of high-cell-density fermentation of Candida utilis on kinetic parameters and the shift to respiro-fermentative metabolism by L. Ordaz; R. López; O. Melchy; M. Torre (pp. 374-378).

Candida utilis NRRLY-900 was grown in a high-cell-density continuous culture without oxygen limitation. Glucose or molasses was used as carbon source at 30 g l^–1 or 100 g (reducing sugars) l^–1. At 30 g glucose l^–1, the dilution rate (D) immediately before the change in respiratory metabolism (D _r) was approximately 0.40 h^–1. At this value of D, the corresponding culture in molasses did not reach the D _r value. When the reducing sugar concentration in the feed was 100 g l^–1, the D _r was 0.15 h^–1 for glucose and 0.3 h^–1 for molasses. When D>D _r, accumulation of ethanol and organic acids occurred, due to physiological changes in C. utilis. The changes observed were a decrease in the biomass yield coefficient per gram of oxygen consumed (Y _O2) and a sudden increase in the specific oxygen consumption rate (qO₂) for each substrate. Therefore, at growth rates above D _r, in a high-cell-density culture, C. utilis acquired a flexible catabolism directed towards alternative fermentation routes. The D at which metabolic changes took place seemed to depend on the nature and concentration of the carbon source. Biomass productivity was higher with molasses than with glucose when the fermenter was operated at high D values.

Metabolic profiles and aprE expression in anaerobic cultures of Bacillus subtilis using nitrate as terminal electron acceptor by J. Espinosa-de-los-Monteros; A. Martinez; F. Valle (pp. 379-384).

Cultures using nitrate as the terminal electron acceptor were conducted in Schaeffer's medium to evaluate the growth performance and metabolic profiles of Bacillus subtilis, and its potential to express the aprE (subtilisin) gene under anoxic conditions. Nitrate was converted to ammonia through nitrite reduction; and different product profiles were observed during the growth phase when nitrate was added at various concentrations (4–24 mM) to Schaeffer's medium containing glucose (4 g l^–1). If nitrate was not limiting, then acetic acid and acetoin were accumulated, suggesting a limitation of reduced cofactors but, if nitrate became limiting, then lactic acid and butanediol were accumulated, suggesting an excess of reduced cofactors. Due to a strong lysis at the onset of the end of the growth phase, sporulation frequency and aprE expression were negligible in anaerobic batch cultures. Fed-batch fermentation allowed the development of a stationary phase through a continuous supply of glucose and nitrate. In this case, sporulation frequency was almost null, but interestingly aprE expression was similar to that found in aerobic cultures.

Selection of Mycobacterium sp. strains with capacity to biotransform high concentrations of β-sitosterol by M. Vidal; J. Becerra; M. Mondaca; M. Silva (pp. 385-389).

In this work, phytosterol-biotransforming strains were selected from Mycobacterium sp., using a high concentration of β-sitosterol. The selection was made by culturing the strains in a medium enriched with 14 g β-sitosterol/l as the unique source of carbon. During 2 months, the bacterial cultures were transferred successively. The extraction of the biotransformation products was made with methanol and ethyl acetate. The qualitative and quantitative analysis was made by means of thin-layer chromatography, gas-liquid chromatography (GLC) and GLC-mass spectrometry. Under these conditions, it was observed that after seven transfers, the strains Mycobacterium sp. MB-3683 and the Mycobacterium fortuitum B-11045 increased their biotransformation capacity from 20% to 64% and from 34% to 55%, respectively. The products in the highest proportion identified for each trial were androstenedione and androstadienedione. The results suggest that the high substrate concentration could be a selective mechanism to obtain strains more efficient in the biotransformation of β-sitosterol into steroidal bases.

Formation of glucoside conjugates during biotransformation of dibenzofuran by Penicillium canescens SBUG-M 1139 by E. Hammer; L. Schoefer; A. Schäfer; K. Hundt; F. Schauer (pp. 390-394).

Penicillium canescens oxidises dibenzofuran (DBF) to produce monohydroxylated derivatives and other more hydrophilic metabolites. These substances are water-soluble but unstable in organic solvents such as ethyl acetate, acetone or dichloromethane. Both extraction with ethyl acetate and enzymatic treatment of the aqueous culture filtrate with β-glucuronidase led to decay of the hydrophilic metabolites and indicated these products to be glycoside conjugates. The glycosyl residue was identified as glucose both by liquid chromatography and by the use of glucose oxidase. The conjugate pattern formed was the same in type and amount, independent of the carbon source used for cultivation of the fungus. Clearly, DBF transformation in P. canescens occurred in two phases: first the conversion to 2-, 3-, and 4-hydroxydibenzofuran (phase I), followed by the formation of the corresponding glucosyl conjugates (phase II). In contrast, 2,3-dihydroxydibenzofuran added to the cultures was transformed by ring cleavage producing a muconic acid-like dead-end product.

Characterisation of extracellular polysaccharides produced by Crypthecodinium cohnii by M. Swaaf; G. Grobben; G. Eggink; T. Rijk; P. Meer; L. Sijtsma (pp. 395-400).

The valuable polyunsaturated fatty acid, docosahexaenoic acid, can be produced by cultivation of the heterotrophic microalga, Crypthecodinium cohnii. During batch growth of C. cohnii on glucose, sea salt and yeast extract for 5 days, so far unreported extracellular polysaccharides were produced. These caused an increased viscosity and a strong drop in the maximum oxygen transfer. The viscosity increased most markedly as cells entered the stationary phase. The polysaccharides varied in size (from 6 kDa to >1,660 kDa) and monomer distribution. A high molecular mass fraction (from 100 kDa to >1,660 kDa) and a medium molecular mass fraction (6–48 kDa) were prepared. The high molecular mass fraction contained (on a molar basis) 71.7% glucose, 13.1% galactose and 3.8% mannose, whereas the medium molecular mass fraction contained 37.7% glucose, 19.8% galactose and 28.1% mannose. Other monomers present in both fractions were fucose, uronic acid and xylose. Monomers were coupled mainly via α-(1–3) links. Increased viscosity due to polysaccharide production complicates the development of commercial, high cell-density processes for the production of docosahexaenoic acid.

New method for exopolysaccharide determination in culture broth using stirred ultrafiltration cells by D. Bergmaier; C. Lacroix; M. Guadalupe Macedo; C. Champagne (pp. 401-406).

A new method to remove simple carbohydrates from culture broth prior to the quantification of exopolysaccharides (EPS) was developed and validated for the EPS-producing strain, Lactobacillus rhamnosus RW-9595M. This method uses ultrafiltration (UF) in stirred cells followed by polysaccharide detection in the retentate by the phenol–sulfuric acid method. The UF method was compared with a conventional method based on ethanol extraction, dialysis, protein removal by trichloroacetic acid (TCA) and freeze-drying. EPS production during pH-controlled batch fermentations in basal minimum medium, whey permeate (WP), and whey permeate supplemented with yeast extract, minerals and Tween-80 (SWP) was determined by the new UF and conventional methods. EPS recovery by the new method ranged from 83% to 104% for EPS added in the concentration range 40–1,500 mg/l in 0.1 M NaCl solution or culture medium. The UF method was rapid (8 h), accurate and simple, and required only a small sample volume (1–5 ml). A very high maximum EPS production was measured in SWP by both the UF and conventional methods (1,718 and 1,755 mg/l).

Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae by H. Atiyeh; Z. Duvnjak (pp. 407-411).

The production of ethanol and enriched fructose syrups from a synthetic medium with various sucrose concentrations using the mutant Saccharomyces cerevisiae ATCC 36858 was investigated. In batch tests, fructose yields were above 90% of theoretical values for the sucrose concentrations between 35 g/l and 257 g/l. The specific growth rates and biomass yields were from 0.218 to 0.128 h^–1 and from 0.160 to 0.075 g biomass/g of glucose and fructose consumed, respectively. Ethanol yields were in the range of 72 to 85% of theoretical value when sucrose concentrations were above 81 g/l. The volumetric ethanol productivity was 2.23 g ethanol/(l h) in a medium containing 216 g/l sucrose. Fructo-oligosaccharides and glycerol were also produced in the process. A maximum fructo-oligosaccharides concentration (up to 9 g/l) was attained in the 257 g/l sucrose medium in the first 7 h of the fermentation. These sugars started to be consumed when the concentrations of sucrose in the media were less than 30% of its initial values. The fructo-oligosaccharides mixture was composed of 6-kestose (61.5%), neokestose (29.7%) and 1-kestose (8.8%). The concentration of glycerol produced in the process was less than 9 g/l. These results will be useful in the production of enriched fructose syrups and ethanol using sucrose-based raw materials.

Molecular fingerprinting of bacterial populations in groundwater and bottled mineral water by T. Dewettinck; W. Hulsbosch; K. Hege; E. Top; W. Verstraete (pp. 412-418).

Monitoring the hygienic quality of drinking waters by determining the concentration of fecal indicators with traditional plate count techniques suffers from important drawbacks. In this work, the potential of PCR-DGGE (polymerase chain reaction – denaturing gradient gel electrophoresis) analysis of 16S rDNA genes to fingerprint the bacterial populations of mineral water and groundwater was investigated. A rapid and simple pretreatment to concentrate and release bacterial DNA prior to PCR was explored. This pretreatment was successful for commercially bottled mineral water. For groundwater, an additional resuscitation step was required to obtain a PCR signal. It was clear that the groundwater under scrutiny contained a more diverse bacterial community than the mineral water. A comparison was made between four kinds of mineral waters and one sample of groundwater using the developed procedures. For each kind of water, bacterial populations cultured on R2A plates were also subjected to PCR-DGGE. Comparison of the fingerprints of the plated samples and the original samples suggested the presence of viable but nonculturable bacteria in the waters. The obtained cluster dendrogram indicated that each kind of water was characterized by a specific molecular fingerprint. The sensitivity of the whole of the procedure was between 10⁴ and 10⁵ cfu ml^–1 as determined using a pure culture of Escherichia coli. The described PCR-DGGE method can constitute the basis of a new and interesting strategy to monitor in a relatively rapid way (less than 24 h) the bacterial quality of waters such as mineral water, groundwater and certain types of reclaimed water.

A GAC biofilm reactor for the continuous degradation of 4-chlorophenol: treatment efficiency and microbial analysis by M. Carvalho; I. Vasconcelos; A. Bull; P. Castro (pp. 419-426).

Using a continuous enrichment technique, a bacterial consortium capable of degrading 4-chlorophenol (4-CP) was obtained from the rhizosphere of Phragmites australis. A granular activated carbon (GAC) biofilm reactor was established using this consortium, and the degradation of 4-CP was investigated under continuous flow operation using a feed of 20–50 mg l^–1 with a hydraulic residence time of 17 min over a 6-month period. Chloride liberation occurred throughout the operation, and the reactor had 4-CP removal efficiencies of 69–100%. Periods of lower performance were attributed to clogging of the column with biomass and the formation of channels. Subsequently, the immobilized biofilm was subjected to a starvation period of 5 months, after which its degradative capacity was still maintained. The microbial consortium was characterized during the continuous flow experiment and dynamic population changes were observed throughout. One isolate recovered from the biofilm was shown to be capable of degrading 4-CP as a sole carbon and energy source.

Atrazine degradation by bioaugmented sediment from constructed wetlands by H. Runes; J. Jenkins; P. Bottomley (pp. 427-432).

The potential to establish pesticide biodegradation in constructed wetland sediment was investigated. Under microcosm conditions, bioaugmentation of sediment with small quantities of an atrazine spill-site soil (1:100 w/w) resulted in the mineralization of 25–30% of ¹⁴C ethyl atrazine (1–10 µg g^–1 sediment) as ¹⁴CO₂under both unsaturated and water-saturated conditions; atrazine and its common metabolites were almost undetectable after 30 days incubation. By comparison, unbioaugmented sediment supplemented with organic amendments (cellulose or cattail leaves) mineralized only 2–3% of ¹⁴C ethyl atrazine, and extractable atrazine and its common metabolites comprised approximately 70% of the original application. The population density of atrazine-degrading microorganisms in unbioaugmented sediment was increased from ~10²/g to 10⁴/g by bioaugmentation (1:100 w/w), and increased by another 60-fold (6.0×10⁵ g^–1) after incubation with 10 µg g^–1 of atrazine. A high population of atrazine degraders (~10⁶ g^–1) and enhanced rates of atrazine mineralization also developed in bioaugmented sediment after incubation in flooded mesocosms planted with cattails (Typha latifolia) and supplemented with atrazine (3.2 mg l^–1, 1 µg g^–1 sediment). In the absence of atrazine, neither the population of atrazine degraders, nor the atrazine mineralizing potential of bioaugmented sediment increased, regardless of the presence or absence of cattails. Bioaugmentation might be a simple method to promote pesticide degradation in nursery run-off channeled through constructed wetlands, if persistence of degraders in the absence of pesticide is not a serious constraint.

Degradation of crude oil by marine cyanobacteria by C. Raghukumar; V. Vipparty; J. David; D. Chandramohan (pp. 433-436).

The marine cyanobacteria Oscillatoria salina Biswas, Plectonema terebrans Bornet et Flahault and Aphanocapsa sp. degraded Bombay High crude oil when grown in artificial seawater nutrients as well as in plain natural seawater. Oil removal was measured by gravimetric and gas chromatographic methods. Around 45–55% of the total fractions of crude oil (containing 50% aliphatics, 31% waxes and bitumin, 14% aromatics and 5% polar compounds) were removed in the presence of these cultures within 10 days. Between 50% and 65% of pure hexadecane (model aliphatic compound) and 20% and 90% of aromatic compounds (anthracene and phenantherene) disappeared within 10 days. Mixed cultures of the three cyanobacterial species removed over 40% of the crude. Additionally, these cultures formed excellent cyanobacterial mats when grown in mixed cultures, and thus have the potential for use in mitigating oil pollution on seashores, either individually or in combination.

Phylogenetic diversity of a SRB-rich marine biofilm by T. Zhang; H. Fang (pp. 437-440).

This study was conducted to characterize the phylogenetic diversity of a corrosive marine biofilm based on 16S rDNA. Results of phylogenetic analysis indicated that, out of the 112 clones developed, 52 clones (46.4%) were affiliated with two families of sulfate-reducing bacteria: Desulfovibrionaceae and Desulfobacteriaceae. Another 44 clones (39.3%) were affiliated with the Clostridiaceae family of low G+C, Gram-positive bacteria. Three clones (2.7%) were closely related to Chlorobium vibrioforme, a green sulfur bacterium.

Biodegradation of radiolabelled synthetic lignin (¹⁴C-DHP) and mechanical pulp in a compost environment by Marja Tuomela; Annele Hatakka; Sanni Raiskila; Minna Vikman; Merja Itävaara (pp. 441-442).

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Applied Microbiology and Biotechnology (v.57, #3)

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Applied Microbiology and Biotechnology (v.57, #3)