Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.45, #1-2)

Triggers for microbial aggregation in activated sludge? by P. Bossier; W. Verstraete (pp. 1-6).

Microbial aggregation into good settling sludge is essential for the well-functioning of activated sludge systems treating waste water. Complete aggregation of all the microbial biomass formed has been proven to be difficult to maintain continuously, resulting in wash-out of suspended solids. This review investigates the possibility that environmental signals could constitute triggers for the induction or stimulation of aggregative physiology.

Influence of detachment, substrate loading and reactor scale on the formation of biofilms in airlift reactors by L. Tijhuis; B. Hijman; M. C. M. Van Loosdrecht; J. J. Heijnen (pp. 7-17).

For a stable and reliable operation of the biofilm airlift suspension reactor (BAS reactor) means to control biomass concentration, biofilm thickness and biofilm morphology are required. For this reason, the influence of applied detachment forces and surface substrate loading on the formation of heterotrophic biofilms in laboratory-scale BAS reactors was studied. Detachment forces were altered by variation of the initial bare carrier concentration or the superficial air velocity. In addition, the dynamics of biofilm formation during start-up of a full scale BAS reactor (300 m³) was monitored and compared with the laboratory-scale start-up (31). This study shows that the biofilm morphology and strength were influenced to a large extent by the surface substrate loading and applied detachment forces. A moderate surface substrate loading and a high detachment force yielded smooth and strong biofilms. The combination of a high surface substrate loading and low detachment forces did lead to rough biofilms, but did not lead to the expected high amount of biomass on the carrier, apparently because of the formation of weaker biofilms. The strength of the biofilms appeared to be related to the detachment forces applied during biofilm formation, in combination with the surface substrate loading. The biofilm morphology and biomass on carrier in the BAS reactor can be controlled using the carrier concentration, substrate loading rate and the superficial air velocity as parameters. The dynamics of biofilm formation during the start-up of a full-scale BAS reactor proved to be similar to heterotrophic biofilm formation in laboratory-scale reactors. This indicates that a model system on the laboratory scale can successfully be applied to predict dynamic phenomena in the full-scale reactor.

Productivity of Spirulina in a strongly curved outdoor tubular photobioreactor by P. Carlozzi; G. Torzillo (pp. 18-23).

The effect of different flow rates (from 0.18 m/s to 0.97 m/s) on the productivity of Spirulina grown outdoors in a strongly curved tubular photo-bioreactor (CTP) was studied. The results were compared to those obtained with a conventional photobioreactor made with straight tubes to form a loop (STP). The cultures were operated at a biomass concentration of 10 g/l dry weight. The productivity of the culture increased by about 39% and 29% in the CTP and STP respectively when the flow rate of the culture was increased from 0.18 m/s to 0.75 m/s. A further increase of the flow rate did not result in any increase of the productivity in either of the photo-bioreactors. The better performance of the culture observed in the CTP was attributed to an intermittent illumination pattern resulting from a secondary flow motion generated in the bends. The power required for the induction of the same flow rate inside the two types of photobioreactor changed significantly. At a Reynolds number of 4000, the power absorbed by the CTP was 25% higher than that required for water recycling in the STP and rose by more than 40% at a Reynolds number of 20000.

Sulfated extracellular polysaccharide production by the halophilic cyanobacterium Aphanocapsa halophytia immobilized on light-diffusing optical fibers by T. Matsunaga; H. Sudo; H. Takemasa; Y. Wachi; N. Nakamura (pp. 24-27).

The cyanobacterium, Aphanocapsa halophytia MN-11, was immobilized in calcium alginate gel and coated on light-diffusing optical fibers (LDOF) for sulfated extracellular polysaccharide production. Results indicated that sulfated extracellular polysaccharide production depends on the number of immobilized cells and the light intensity. In addition, the production rate reached 116.0 mg (mg dry cells)⁻¹ day⁻¹ when the cells that were immobilized on LDOF were incubated under a light intensity of 1380 cd sr m⁻² at a cell concentration of 1.0 × 10⁸ cells/cm³ gel. Cells immobilized on LDOF produced about ten times more sulfated extracellular polysaccharide than those immobilized in calcium alginate beads only (11.7 mg(mg dry cells)⁻¹ Day⁻¹).

Citric acid accumulation by cycloheximide-sensitive mutant strains of Aspergillus niger by S. Rugsaseel; K. Kirimura; S. Usami (pp. 28-35).

Mutants having impaired protein synthesis, that is cycloheximide-sensitive mutants of a citric-acid-hyper-accumulating strain, were induced from Aspergillus niger WU-2223L. Selection was on the basis of a presumption that the mutants should be more sensitive to cycloheximide than WU-2223L. In shake culture without methanol as a promotor substance, seven mutants accumulated approximately 1.8–3.5 times as much citric acid as WU-2223L. The best mutant, CHM I-C3, accumulated 69.4 mg citric acid/ml from 120 mg glucose/ml in shake culture without methanol, this amount being 1.1 times the amount accumulated by WU-2223L with methanol. Furthermore, under the conditions without methanol the mutants appeared to be more efficient than WU-2223L in employing the consumed glucose for the accumulation of citric acid. It was also confirmed that CHM I-C3 exhibited a significantly increased level of intracellular NH ₄ ⁺ accumulation. The addition of 2% (v/v) methanol or 20 μg cycloheximide/ml to the medium caused a remarkable increase of citric acid accumulation by WU-2223L: about 3.1 and 2.4 times respectively. However, the addition of these substances produced negative effects on citric acid accumulation by the mutants. With 2% (v/v) methanol, WU-2223L showed a remarkably decreased level of protein accumulation but a substantially increased level of intracellular NH ₄ ⁺ accumulation. However, these phenomena were also observed in CHM I-C3 without methanol. These results indicate that the intracellular circumstances of the cycloheximide-sensitive mutants without methanol were similar to those of WU-2223L with methanol, and that the impairment of protein synthesis contributed to increased citric acid accumulation by the mutants in the absence of methanol.

Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: relationship between production of the lantibiotic and lactate and cell growth by H. Matsusaki; N. Endo; K. Sonomoto; A. Ishizaki (pp. 36-40).

The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied. Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail. Production of nisin Z was optimal at 30°C and in the pH range 5.0–5.5. The addition of Ca²⁺ to the medium showed a stimulating effect on the production of nisin Z. A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl₂. It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production.

Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations by A. F. Mayer; W. -D. Deckwer (pp. 41-46).

Clavulanic acid (CA) was produced by Streptomyces clavuligerus in medium containing glycerol and soy meal or soy meal extract. With regard to growth and CA productivity, the microorganism showed significant differences if solid soy meal as such or its extract were applied as the major nitrogen source. If the extract is used, growth and CA production take place simultaneously and in the stationary phase the CA concentration is stagnant or reduces. If soy meal is used, growth is threefold faster and CA is only generated in the stationary phase. In the case of using the soy meal extract, the decrease of the CA concentration is mainly due to decomposition or re-metabolisation of CA in the presence of the microorganism. This conclusion is supported by in vivo and in vitro data on CA decomposition.

Influence of iron, phosphate and methyl viologen on glycerol fermentation of Clostridium butyricum by A. Reimann; H. Biebl; W. -D. Deckwer (pp. 47-50).

The effect of methyl viologen addition, and iron and phosphate limitation on product distribution during glycerol fermentation of Clostridium butyricum DSM 5431 was investigated in continuous culture. Special attention was paid to the gaseous products H₂ and CO₂, which were measured on-line. In all three cases, an increased yield of 1,3-propanediol linked to a decreased hydrogen release was observed, indicating that a higher proportion of electrons was channelled from reduced ferredoxin towards NADH₂ production. The specific substrate consumption rates and the specific production rates revealed that this increase in propanediol yield was not obtained at the expense of glycolysis products but by an increased substrate conversion (overflow metabolism). The acetate/ butyrate ratio during glycerol fermentation was essentially influenced by the availability of iron. It was substantially increased when the culture turned from iron excess to iron-limited conditions. Therefore iron limitation proved to be a suitable means to achieve high 1,3-propanediol yields and to reduce butyrate formation.

Enzymatic preparation of optically active 3-trimethylsilylalanine by H. Yamanaka; T. Fukui; T. Kawamoto; A. Tanaka (pp. 51-55).

We constructed an efficient system for preparing optically active 3-trimethylsilylalanine (TMS-Ala) by kinetic resolution with acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC 3.5.1.14). Racemic TMS-Ala was chemically synthesized and acetylated. Enantioselective deacetylation of N-acetyl-dl-TMS-Ala with acylase I from porcine kidney or from Aspergillus melleus was then attempted. Both enzymes could catalyze the deacetylation of N-acetyl-dl-TMS-Ala, and the porcine enzyme was found to have much higher activity than the enzyme from A. melleus. The optimum pH of the porcine-acylase-catalyzed reaction was 7.5, and the addition of 0.5 mM Co²⁺ accelerated the reaction. Optically pure l-TMS-Ala (> 99% enantiomeric excess, ee) was obtained in 72% yield under the optimized conditions. Furthermore, highly optically pure d-TMS-Ala (96% ee) could also be obtained in 76% yield by chemically hydrolyzing the residual substrate.

Expression and substrate specificity of the toluene dioxygenase of Pseudomonas putida NCIMB 11767 by S. C. Heald; R. O. Jenkins (pp. 56-62).

Pseudomonas putida NCIMB 11767 oxidized phenol, monochlorophenols, several dichlorophenols and a range of alkylbenzenes (C₁–C₆) via an inducible toluene dioxygenase enzyme system. Biphenyl and naphthalene were also oxidized by this enzyme. Growth on toluene and phenol induced the meta-ring - fission enzyme, catechol 2,3-oxygenase, whereas growth on benzoate, which did not require expression of toluene dioxygenase, induced the ortho-ring-cleavage enzyme, catechol 1,2-oxygenase. Monochlorobenzoate isomers and 2,3,5-trichlorophenol were gratuitous inducers of toluene dioxygenase, whereas 3,4-dichlorophenol was a fortuitous oxidation substrate of the enzyme. The organism also grew on 2,4- and 2,5-dichloro isomers of both phenol and benzoate, on 2,3,4-trichlorophenol and on 1-phenylheptane. During growth on toluene in nitrogen-limited chemostat culture, expression of both toluene dioxygenase and catechol 2,3-oxygenase was positively correlated with increase in specific growth rate (0.11–0.74 h⁻¹), whereas the biomass yield coefficient decreased. At optimal dilution rates, the predicted performance of a l-m³ bioreactor supplied with 1 g nitrogen l⁻¹ for removal of toluene was 57 g day⁻¹ and for removal of trichloroethylene was 3.4 g day⁻¹. The work highlights the oxidative versatility of this bacterium with respect to substituted hydrocarbons and shows how growth rate influences the production of competent cells for potential use as bioremediation catalysts.

Purification of alkaline proteases from a Bacillus strain and their possible interrelationship by T. Kobayashi; Y. Hakamada; J. Hitomi; K. Koike; S. Ito (pp. 63-71).

Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE). The major protease, designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism. H protease had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein was observed at pH 11.0 and at 55°C. N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE, although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and at 60°C. The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease. The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values and at 5°C. M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease at pH 8 or below but not at pH 11. N protease appeared to be the autolytic product of the M and H proteases.

Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides by K. N. Faber; S. Westra; H. R. Waterham; I. Keizer-Gunnink; W. Harder; G. AB; M. Veenhuis (pp. 72-79).

We describe the synthesis and purification of two functional peptides, namely human insulin-like growth factor II (IGF-II) and Xenopus laevis magainin II in Hansenula polymorpha after their synthesis as hybrid proteins fused to the C terminus of endogenous amine oxidase. The hybrid genes, placed under control of the H. polymorpha alcohol oxidase promoter (P_AOX were integrated into the genomic alcohol oxidase locus, yielding stable production strains. High-level synthesis of the fusion proteins, exceeding 20% of total cellular protein, was obtained when the transformed strains were grown in methanol-limited chemostat cultures; when expressed by itself, i.e. in the absence of the amine oxidase gene, IGF-II could not be recovered from crude cell extracts, probably as a result of rapid proteolytic degradation. Accumulation in peroxisomes did not significantly affect the IGF-II protein stability when expressed in the absence of the carrier protein. Apparently, fusion to the large (±78 kDa) amine oxidase carrier particularly stabilizes the peptides and prevents them from proteolysis. After partial purification, the fusion partners were readily separated by factor Xa treatment.

Cloning and sequence analysis of the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus by K. Ikeda; T. Masujima; K. Suzuki; M. Sugiyama (pp. 80-85).

Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin-like pigment. An operon, designated mel, containing a gene that encodes tyrosinase, which is involved in the synthesis of melanin pigment, was cloned from the chromosomal DNA of the microorganism into the high-copy plasmid pAK114 and expressed in S. lividans. The tyrosinase activity of the transformed cells was at approximately a 110-fold higher level than that of the same host carrying the plasmid pIJ702, which has the same replication origin as pAK114 and carries the mel operon from S. antibioticus. The sequence analysis of the S. castaneoglobisporus mel operon revealed that an open-reading frame consisting of 378 base pairs(bp), designated ORF378, was found upstream of the tyrosinase gene (TYRC) consisting of 819 bp. In the present study, we constructed a chimeric mel operon consisting of ORF378 from S. castaneoglobisporus and the tyrosinase gene (TYRA) from S. antibioticus. The chimeric mel operon or the S. antibioticus mel operon, which consists of ORF438 and TYRA, expressed the tyrosinase activity in Escherichia coli intracellularly when located under the control of lacZ promoter, and the tyrosinase activity from the former was at a 30-fold higher level than that from the latter. This suggests that the gene contributing to the high expression of the tyrosinase activity in S. castaneoglobisporus is ORF378, rather than TYRC.

Cloning, sequencing and overexpression in Escherichia coli of a xylanase gene, xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor by P. P. Dwivedi; M. D. Gibbs; D. J. Saul; P. L. Bergquist (pp. 86-93).

A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in λZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selezctive polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70°C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp.

Efficient production of Thermus protease aqualysin I in Escherichia coli: effects of cloned gene structure and two-stage culture by S. Sakamoto; I. Terada; Y. -C. Lee; K. Uehara; H. Matsuzawa; M. Iijima (pp. 94-101).

The DNA sequence encoding Thermus protease aqualysin I was inserted downstream from a bacteriophage T7 promoter in an expression vector. In the T7 expression system, using a strain lacking an F′ episome, aqualysin I was produced in soluble form without chemical induction. The deletions of part (30 amino acid residues) or all (105 residues) of the C-terminal pro-sequence from the C terminus significantly affected both cellular growth and the production of the enzyme. Complete deletion adversely affected both. In contrast, the 30-residue deletion markedly improved productivity by approximately four times compared to non-deletion, and shortened the time needed for the activation of a precursor to active enzyme. The concentration of inducer isopropyl β-D-thiogalactopyrano-side (IPTG) was varied to examine its effects, and it was found that a low concentration of IPTG improved aqualysin I production. To avoid the inhibitory effects of acetic acid accumulation in the culture medium, the use of other carbon sources besides glucose was examined. When cells were cultivated with glycerol, the acetic acid level remained relatively low, and both good cellular growth and a high level of production were attained. The aqualysin I

Expression of the Arxula adeninivorans glucoamylase gene in Kluyveromyces lactis by D. M. Bui; I. Kunze; C. Horstmann; T. Schmidt; K. D. Breunig; G. Kunze (pp. 102-106).

The glucoamylase gene of the yeast Arxula adeninivorans was expressed in Kluyveromyces lactis by using the GAP promoter from Saccharomyces cerevisiae and a multicopy plasmid vector. The transformants secreted 90.1% of the synthesized glucoamylase into the culture medium. The secreted glucoamylase activities are about 20 times higher in comparison to those of Saccharomyces cerevisiae transformants using the same promoter. Secreted glucoamylase possesses identical N-terminal amino acid sequences to those secreted by A. adeninivorans showing that cleavage of the N-terminal signal peptide takes place at the same site. Biochemical characteristics of glucoamylase expressed by K. lactis and A. adeninivorans are very similar.

Effect of ilvBN-encoded α-acetolactate synthase expression on diacetyl production in Lactococcus lactis by K. H. Benson; J. -J. Godon; P. Renault; H. G. Griffin; M. J. Gasson (pp. 107-111).

Conversion of pyruvate to α-acetolactate, which is broken down to diacetyl and acetoin, can be catalysed by two α-acetolactate synthases in Lactococcus lactis. The enzyme encoded by the als gene (Als) has previously been shown to have a low affinity for pyruvate, which limits the formation of diacetyl. In this study we have expressed from a plasmid the ilvBN genes, which encode the other α-acetolactate synthase (IlvBN). This plasmid-directed enzyme expression provided up to 3.6-fold increased product formation in the L. lactis MG1363 and IL1403 backgrounds. Plasmid-based expression of the ilvBN genes, in an IL1403 derivative from which the leu.ilv.ald and flanking genes had been deleted, yielded up to 0.1 mM diacetyl where-as the host strain produced none. In addition, IlvBN, with a K _m value of 8.3 mM, was shown to have a greater affinity for pyruvate than does Als.

Characterization of Escherichia coli expressing an Lpp’OmpA(46-159)-PhoA fusion protein localized in the outer membrane by C. Stathopoulos; G. Georgiou; C. F. Earhart (pp. 112-119).

The Lpp′OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusions between Lpp′OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp′OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp′OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (β-lactamase, alkaline phosphatase, Cex cellulase, Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp′OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.

Temperature-regulated expression of the tac/lacI system for overproduction of a fungal xylanase in Escherichia coli by G. -P. Xue; J. S. Johnson; D. J. Smyth; L. M. Dierens; X. Wang; G. D. Simpson; K. S. Gobius; J. H. Aylward (pp. 120-126).

Temperature-regulated expression of recombinant proteins in the tac promoter (P_tac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the P_tac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42°C were about 4.5 times higher than those of the cells grown at 23°C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl β-d-thiogalactopyranoside. The xylanase expression level in the temperature-regulated P_tac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacI^q, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the λP l system, the P_tac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best λP l-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated P_tac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperaturemodulated P_tac system can be used for overproduction of some non-toxic recombinant proteins.

Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum by T. R. Klapatch; A. L. Demain; L. R. Lynd (pp. 127-131).

Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55°C. The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling > 100 kb, insuring that most potential restriction sequences have been exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5′ GATC 3′. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam⁺ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam⁻ phenotype.

Characterisation of a complex restriction/modification system detected in a Bifidobacterium longum strain by A. Hartke; A. Benachour; P. Boutibonnes; Y. Auffray (pp. 132-136).

Two type-II restriction endonucleases, BloI and BloII, have been detected in a Bifidobacterium longum strain. BloI is influenced by dam methylation: it cleaves dam ⁻ but not dam ⁺ DNA. It shows a temperature and pH optimum of 45°C and pH 7.5. Restriction analysis and cloning experiments showed that the recognition sequence is RGATCY and that the enzyme cuts 5′ to the guanine residue. It is an isoschizomer of commercial enzymes, Bst YI and XhoII. The second activity is not inhibited by dam methylation. It has a temperature optimum between 25°C and 30°C and shows a broad pH optimum between 4.5 and 7.0. The activity is thermolabile and can be heat-killed by a 5 min incubation at 60°C. Cloning and sequencing experiments revealed that its recognition sequence is CTGCAG and that it cuts 5′ to the second guanine residue in the sequence. This enzyme is the first described isoschizomer of PstI.

Reduction of RNA and DNA in Methylococcus capsulatus by endogenous nucleases by J. Larsen; L. Joergensen (pp. 137-140).

A method for reducing RNA and DNA in the bacterium Methylococcus capsulatus (Bath) has been developed. Endogenous RNase and DNase were activated by a 10 s heat shock at 90°C. Cells were then incubated at 60°C for 20 min to allow degradation of the nucleic acids. The optimum pH for the process was 7.0. The protein loss was less than 10% and occurred during the initial heat shock. No protein loss was found during incubation. The total dry-weight loss in connection with an 80% reduction of the nucleic acid content was 20%–25%, giving a final product with a raw protein content of approximately 75%. Reduction of both RNA and DNA was inhibited by CuSO₄ and ZnSO₄. DNA reduction was stimulated by other minerals. Optimal stimulation was found at 1 mM FeSO₄. Reduction of RNA was not increased by any of the minerals tested.

Cloning and analysis of a gene from Streptomyces lividans 66 encoding a novel secreted protease exhibiting homology to subtilisin BPN′ by M. J. Butler; J. S. Aphale; C. Binnie; M. A. DiZonno; P. Krygsman; G. Soltes; E. Walczyk; L. T. Malek (pp. 141-147).

Amino-terminal degradation has been observed for many of the secreted heterologous proteins produced by S. lividans 66. We, therefore, set out to characterize the relevant proteinases and their genes. A tripeptide chromogenic substrate was used to identify a gene that was shown to encode a secreted protein which removed tripeptides from the amino terminus of extracellular proteins (tripeptidyl aminopeptidase, Tap; Butler et al. 1995). This activity was removed by a homologous gene deletion replacement and the ability of the S. lividans strain to remove N-terminal tripeptides was greatly reduced, but still significant. When the tap-deleted strain was used as a host for the rescreening of a S. lividans 66 genomic DNA library, a number of other genes encoding proteases with aminopeptidase activities were discovered. One clone (P5-4) produced a 45-kDa secreted protein (Ssp), which showed activity against Ala-Pro-Ala-β-naphthylamide (APA-βNH-Nap) substrate. Further analysis of the cloned DNA showed an open-reading frame encoding a protein larger than 45 kDa. Direct Edman degradation of the secreted protein confirmed that it was encoded within the cloned DNA and probably processed from a larger precursor. Protein sequence analysis revealed a striking homology to subtilisin BPN′ in three regions around the active-site residues suggesting that the protein is a serine protease. As expected, the protease activity was inhibited by phenylmethylsulphonyl fluoride. Mutant strains with most of the ssp gene deleted exhibited reduced activity against APA-βNH-Nap substrate compared to their non-deleted parental strains.

Killing of microorganisms by pulsed electric fields by T. Grahl; H. Märkl (pp. 148-157).

Lethal effects of pulsed electric fields (PEF) on suspensions of various bacteria, yeast, and spores in buffer solutions and liquid foodstuffs were examined. Living-cell counts of vegetative cell types were reduced by PEF treatment by up to more than four orders of magnitude (> 99.99%). On the other hand, endoand ascospores were not inactivated or killed to any great extent. The killing of vegetative cell types depends on the electrical field strength of the pulses and on the treatment time (the product of the pulse number and the decay time constant of the pulses). For each cell type, a specific critical electric field strength (E _c) and a specific critical treatment time (t _c) were determined. Above these critical values, the fractions of surviving cells were reduced drastically. The “limits” E _c and t _c depend on the cell characteristics as well as on the type of medium in which the cells are suspended. Especially in acid media living-cell counts were sufficiently decreased at very low energy inputs. In addition to the inactivation of microorganisms, the effect of PEF on food components such as whey proteins, enzymes and vitamins, and on the taste of foodstuffs was studied. The degree of destruction of these food components by PEF was very low or negligible. Moreover, no significant deterioration of the taste of foodstuffs was detected after PEF treatment. Disintegration of cells by PEF treatment in order to harvest intracellular products was also studied. Yeast cells, suspended in buffer solution, were not disintegrated by electric pulses. Hence, PEF treatment is an excellent process for inactivation of microorganisms in acid and in thermosensive media, but not for complete disintegration of microbial cells.

Application of a microtitre reader system to the screening of inulinase nulinase-producing yeasts by G. C. Passador-Gurgel; S. A. Furlan; J. K. Melier; R. Jonas (pp. 158-161).

Fourteen strains of yeast from genera Kluyveromyces, Candida, Debaryomyces and Schizosaccharomyces were investigated for inulinase production. In the first stage, the microtitre reader system SLT was used for the determination of enzyme activity and the evaluation of cellular growth. Different culture conditions were tested and four strains of Kluyveromyces were selected on the basis of enzyme activity and growth capacity at low pH and high temperature: K. marxianus CBS 6397, DSM 70792, ATCC 36907 and IZ 619. These strains were tested in greater volume using pH 4.0, 45°C and inulin (10 g/l) as selection conditions. On the basis of results obtained, the strain K. marxianus ATCC 36907 was selected for inulinase production. Enzyme stability at low pH (4.0) as well as high temperature (50°C) for 10, 30 and 60 min was also evaluated, but no significant difference in enzyme activity was observed. It could be demonstrated that the microtitre reader system is an excellent method for the screening of microorganisms.

Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species by S. Arino; R. Marchal; J. -P. Vandecasteele (pp. 162-168).

A glycolipid-producing bacterium, Pseudomonas aeruginosa GL1, was isolated from the soil contaminated with polycyclic aromatic hydrocarbons (PAH) from a manufactured gas plant. The glycolipid produced was characterized in detail by chromatographic procedures as a mixture of four rhamnolipids, consisting of different associations of rhamnose and hydroxy fatty acids: the main component was monorhamnosyl di-3-hydroxydecanoic acid. The rhamnolipid composition presented marked analogies with a defined part of P. aeruginosa outer membrane lipopolysaccharides (lipopolysaccharide band A). Rhamnolipid production was stimulated under conditions of nitrogen limitation. Glycerol yielded higher productions than did hydrophobic carbon sources. Cell hydrophobicity decreased during growth on glycerol and on n-hexadecane whereas glycolipid production increased. P. aeruginosa GL1 was found to be unable to grow on a variety of 2, 3 and 4 cycle PAH. However, it was shown to persist after at least 12 subcultures in a bacterial population growing on a mixture of pure PAH, suggesting a physiological role for rhamnolipid as a means to enhance PAH availability in a mutualistic PAH-degrading bacterial community.

Quantitative measurement of sulphur formation by steady-state and transient-state continuous cultures of autotrophic Thiobacillus species by G. C. Stefess; R. A. M. Torremans; R. de Schrijver; L. A. Robertson; J. G. Kuenen (pp. 169-175).

Sulphur formation by the obligately chemolithoautotrophic Thiobacillus o and Thiobacillus neapolitanus was studied in aerobic, substrate-limited continuous cultures. The performance of transient-state and steady-state cultures was compared using different methods for measuring sulphur production. Below a dilution rate (D) of 0.3 h⁻¹ (at 50% air saturation), sulphate-producing steady states were obtained, and cultures grown with sulphide or thiosulphate (at D = 0.06 h⁻¹) showed similar characteristics (e.g. cell yields, oxidation capacities and CO₂-fixation capacities). Elemental sulphur was a major product above D = 0.3 h⁻¹, but steady states were difficult to achieve, because of adherence of sulphur to the fermentor surfaces and the accumulation of sulphide. These problems could be circumvented using transient-state experiments of 1 h. It was then found that elemental sulphur was formed under oxygen limitation or at high substrate load. The rates of sulphur formation obtained by sulphur analysis agreed with the values calculated from stoichiometric balances. Sulphide and thiosulphate proved to be equivalent substrates for both Thiobacillus species during elemental sulphur formation under the conditions tested. It is concluded that transient-state cultures of thiobacilli, pregrown as sulphate-producing steady-state cultures, provide experimental conditions for the quantitative assessment of sulphur formation from (labile) sulphide and from thiosulphate.

Hyperinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens by M. Kobayashi; T. Fujita; S. Shimizu (pp. 176-181).

We investigated the optimum conditions for the formation of nitrile hydratase (NHase), which acts on indole-3-acetonitrile, in Agrobacterium tumefaciens. Good inducers for enzyme formation have been found to be roughly classified into three representative types of amides such as pivalamide, crotonamide and e-caprolactam. When the strain was cultivated in the optimum culture medium containing ε-caprolactam as an inducer, in particular, the specific activity of NHase in the culture was 13 000 times higher than that without addition of amides, nitriles or acids. In this case, NHase formed accounted for 12% of the total cellular soluble protein. The purified NHase did not act on ε-caprolactam, and ε-caprolactam was not degraded during the cultivation by the strain, suggesting that ε-caprolactam seems to keep driving the NHase induction mechanism.

Rhizobium etli cytochrome mutants with derepressed expression of cytochrome terminal oxidases and enhanced symbiotic nitrogen accumulation by J. Miranda; J. Membrillo-Hernández; M. L. Tabche; M. Soberón (pp. 182-188).

A method to isolate mutants with derepressed expression of cytochrome oxidases and better symbiotic performance is presented. A mutant of Rhizobium etli, CFN030, isolated by its azide-resistant phenotype, was obtained by transposon Tn5 -mob mutagenesis. This mutant has a derepressed expression of cytochrome aa₃, higher respiratory activities when cultured microaerobically and an improved symbiotic nitrogen fixation capacity. This phenotype was similar to the previously described mutant CFN037, which was isolated by its increased capacity to oxidize N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) [Soberón M et al. (1990) J Bacteriol 172:1676–1680]. We show here that although both mutants have a similar symbiotic phenotype, they are affected in different genes. Strain CFN030 has the Tn5 inserted in the chromosome while in strain CFN037 the transposon was located in plasmid b. Cytochrome spectral analysis of both mutant strains in the post-exponential phase of growth, showed the expression of an additional terminal oxidase (cbb₃) that is not expressed in the wild-type strain.

Mutational analysis of the Streptomyces scabies esterase signal peptide by V. A. Hale; J. L. Schottel (pp. 189-198).

Ten site-directed mutations affecting the predicted 39-amino-acid signal peptide of the Streptomyces scabies esterase were used to examine start-codon usage and esterase secretion in S. lividans. The first of two in-frame AUG codons was preferred for translation initiation. Removal of 2 of the 4 positively charged amino acids at the amino terminus of the signal peptide reduced esterase expression more than 100-fold; however, deletion of all 4 charged residues reduced expression by only 2- to 5-fold. Deletion of 4 or 8 amino acids from the hydrophobic core of the signal peptide reduced esterase production more than 200-fold, and a signal peptide processing site deletion completely disrupted esterase expression. For all constructs in which a mutation in the signal sequence decreased esterase production, esterase mRNA levels were also reduced, suggesting that a defect in secretion or processing affected esterase transcript abundance.

Transition rate kinetics from ethanol oxidation to glucose utilisation within a structured model of baker’s yeast by P. Dantigny; M. Gruber (pp. 199-203).

The transition rate kinetics from ethanol oxidation to glucose utilisation, within a structured model of baker’s yeast, described previously, were experimentally identified. The shift in metabolism has been assessed through glucose pulses during batch growth on ethanol. The influence of glucose concentration (between 0.25 g l⁻¹ and 0.90 g l⁻¹) and initial biomass concentration (between 0.61 g l⁻¹ and 1.44 g l⁻¹) on the transition rate was determined. The transition rate can not be described by a first-order saturation-type kinetics with respect to glucose only. A corrective term, which takes into account biomass concentration should be included.

Regulation of spiramycin synthesis in Streptomyces ambofaciens: effects of glucose and inorganic phosphate by A. Lounès; A. Lebrihi; C. Benslimane; G. Lefebvre; P. Germain (pp. 204-211).

The production of the 16-membered macrolide antibiotic, spiramycin, in Streptomyces ambofaciens is inhibited by glucose, 2-deoxyglucose and inorganic phosphate. The role of intracellular ATP content and phosphorylated metabolites as common regulating signals of both glucose and phosphate inhibitory effects is discussed. Two enzymatic targets of the effect of phosphate on spiramycin biosynthesis were studied. Valine dehydrogenase, the first enzyme of valine catabolism (supplier of aglycone spiramycin precursors), and alkaline phosphatase, which cleaves phosphorylated intermediates, were repressed in the presence of excess phosphate.

Toxicity of arsenic during high temperature bioleaching of gold-bearing arsenical pyrite by K. B. Hallberg; H. M. Sehlin; E. B. Lindström (pp. 212-216).

A moderately thermophilic mixed culture, MT, and the thermophilic Sulfolobus acidocaldarius strain BC were studied for their response to arsenic in a defined medium and also in media containing a pyrite and an arsenical pyrite flotation concentrate. In defined medium, the individual constituents of the MT culture exhibited a high tolerance to arsenite and arsenate compared to S. acidocaldarius strain BC. When grown on increasing concentrations of the pyrite flotation concentrate, both cultures had similar specific leaching rates over the various concentrations of the mineral substrate. In contrast, S. acidocaldarius strain BC exhibited a decreasing specific leaching rate when grown on the arsenical pyrite while the MT culture was not affected. In addition, arsenic added to cultures of S. acidocaldarius strain BC growing with pyrite as a growth substrate inhibited further growth, while added arsenic had no effect on the MT culture growing on the pyrite. These data indicate that the moderately thermophilic, arsenic-resistant MT culture was able to leach arsenical pyrite more efficiently than was the S. acidocaldarius strain BC culture at high concentrations of the mineral. This emphasizes the fact that proper culture selection is an important parameter when developing commercial processes involving arsenic-containing minerals.

Simultaneous utilization of pyridine and fructose by Rhodococcus opacus UFZ B 408 without an external nitrogen source by U. Brinkmann; W. Babel (pp. 217-223).

A bacterium classified as Rhodococcus opacus, which is able to use pyridine (a potentially growth-inhibiting substrate) as its sole source of carbon, energy and nitrogen, was isolated. In a carbon-limited chemostat culture, the kinetics was determined for growth on both pyridine and a mixture of pyridine and fructose (9 mM/22.15 mM). With growth on pyridine, stable steady states were achieved up to dilution rates of about 0.1 h⁻¹. A further increase in the dilution rate resulted in the progressive accumulation of pyridine in the culture liquid and the cells were washed out. The maximum specific growth rate (μ_max = 0.23 h⁻¹) and the K_S value (0.22 mM) for growth on pyridine were determined from the residual pyridine concentrations measured within the range of stable steady states. With growth on the substrate mixture, the specific pyridine consumption rates and the residual pyridine concentrations were lower at similar dilution rates than with growth on pyridine alone, and stable steady states were established at dilution rates of up to 0.13 h⁻¹. The maximum pyridine degradation rate was enhanced to 270 mg pyridine l⁻¹ h⁻¹ compared to 210 mg pyridine l⁻¹ h⁻¹ with growth on pyridine as a single substrate. An external nitrogen source did not need to be added in the case of growth on the substrate mixture. Fructose was assimilated by means of ammonium released from pyridine. Analysis of the nitrogen balance furnished proof that pyridine is an energy-deficient substrate; pyridine was assimilated and dissimilated at a ratio of 1 mol/0.67 mol respectively. The resulting yield coefficient was about 0.55 g dry weight/g pyridine. Moreover, it was demonstrated that, in regard to the biologically usable energy, 1 mol pyridine corresponds to 0.43 mol fructose.

Effect of pH on cell viability and product yields in d-xylose fermentations by Candida shehatae by J. R. Kastner; R. S. Roberts; W. J. Jones (pp. 224-228).

Candida shehatae were sequentially subjected to aerobic conditions for cellular growth, followed by anaerobic conditions for ethanol production from d-xylose at pH 2.5,4.5 and 6.0. Ethanol yields increased from 0.25 g/g to 0.37 g/g and xylitol yields decreased from 0.33 g/g to 0.1 g/g as the pH was increased from 2.5 to 6.0. Cell viability, measured by plate counts and methylene blue staining, decreased in all of the fermentations, following the switch from aerobic to anaerobic conditions. However, pH 6.0 was shown to extend cell viability and increase the final ethanol concentration from 45 g/l to 55 g/l, compared to the yield at pH 4.5.

Influence of nitrate starvation on nitrite accumulation during denitrification byPseudomonas stutzeri by B. Xu; S. -O. Enfors (pp. 229-235).

Anaerobically denitrifyingPseudomonas stutzeri may transiently accumulate from 0% to 100% of nitrate as nitrite, depending on the nitrate availability during the preceding 24 h. The variations of transient nitrite level were related to the length of previous nitrate starvation. Cells harvested in a late anaerobic growth phase did not accumulate any nitrite during denitrification. Five hours of nitrate starvation caused about 40% (mol/mol) nitrite formation from the further added nitrate and 16 h nearly 80%. Concomitant with this, whereas the nitrate reduction capacity was not significantly affected, the initial nitrite reduction capacity was decreased. These results suggested that nitrate starvation caused a rapid loss of the originally produced nitrite-reducing capacity in the preculture. The lost capacity could be regenerated when nitrate or nitrite was resupplied to the cells. To investigate the nature of the restoration of nitrite-reducing capacity, chloramphenicol was used. The presence of chloramphenicol at 83 μg/ml entirely stopped this restoration, which was otherwise observed in all instances. This indicated that the recovery of nitrite-reducing activity required de novo protein synthesis, which was further confirmed by Western immunoblot assay of cd₁ nitrite reductase.

Effect of pH on transfructosylation and hydrolysis by β-fructofuranosidase from Aspergillus oryzae by M. Kurakake; T. Onoue; T. Komaki (pp. 236-239).

β-Fructofuranosidase was purified from commercial alkaline protease (Aspergillus oryzae origin). The optimal pH of its transfructosylating activity was more alkaline (pH 8) than that of its hydrolyzing activity (pH 5). In the case of a 24-h reaction with sucrose, the hydrolysis and transfructosylation reaction were optimal at pH 4–5 and pH 8, respectively. In the reaction at pH 8 1-kestose and nystose were the main fructooligosaccharides produced. The transfer ratio was hardly different between pH 5 and pH 8 early in the reaction, but the transfer products (1-kestose and nystose) were decreased at pH 5 as the reaction proceeded because of their hydrolysis.

Detection of endogenous β-glucuronidase activity in Aspergillus niger by T. E. Gottschalk; J. E. Nielsen; P. Rasmussen (pp. 240-244).

An endogenous β-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous β-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor d-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uidA β-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7–8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous β-glucuronidase activity, was achieved by staining at this pH.

The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant by V. Zverlov; K. Piotukh; O. Dakhova; G. Velikodvorskaya; R. Borriss (pp. 245-247).

The nucleotide sequence of the xynA gene, encoding extracellular xylanase A of Thermotoga neapolitana, was determined. The xynA gene was 3264 base pairs (bp) long and encoded a putative polypeptide of 1055 amino acids. Three different domains were identified by sequence comparison and functional analysis of proteins with N- and/or C-terminal deletions. The core domain displayed significant homology to members of the glycosyl hydrolase family 10. N- and C-terminal domains were dispensable for enzymatic activity and seemed to be responsible for thermostability and cellulose binding, respectively. The intact gene and its truncated variants were expressed in Escherichia coli and purified for biochemical characterization. The enzyme was shown to act as an endo-1,4-β-xylanase, but minor activities against lichenan, barley glucan, methylumbelliferyl cellobioside and p-nitrophenyl xyloside were also detected. The specific activity and pH and temperature optima for hydrolysis of oat xylan were 111.3 U·mg⁻¹, 5.5 and 102°C, respectively. The endoxylanase was stable at 90°C and retained 50% activity when incubated for 2 h at 100°C.

Trichloroethylene degradation and mineralization by pseudomonads and Methylosinus trichosporium OB3b by A. K. Sun; T. K. Wood (pp. 248-256).

To examine the trichloroethylene (C₂HCl₃)-degrading capability of five microorganisms, the maximum rate, extent, and degree of C₂HCl₃ mineralization were evaluated for Pseudomonas cepacia G4, Pseudomonas cepacia G4 PR1, Pseudomonas mendocina KR1, Pseudomonas putida F1, and Methylosinus trichosporium OB3b using growth conditions commonly reported in the literature for expression of oxygenases responsible for C₂HCl₃ degradation. By varying the C₂HCl₃ concentration from 5 μM to 75 μM, V_max and K_m values for C₂HCl₃ degradation were calculated as 9 nmol/(min mg protein) and 4 μM for P. cepacia G4, 18 nmol/(min mg protein) and 29 μM for P. cepacia G4 PR1, 20 nmol/(min mg protein) and 10 μM for P. mendocina KR1, and 8 nmol/(min mg protein) and 5 μM for P. putida F1. This is the first report of these Michaelis-Menten parameters for P. mendocina KR1, P. putida F1, and P. cepacia G4 PR1. At 75 μM, the extent of C₂HCl₃ that was degraded after 6 h of incubation with resting cells was 61%–98%; the highest degradation being achieved by toluene-induced P. mendocina KR1. The extent of C₂HCl₃ mineralization in 6 h (as indicated by concentration of chloride ion) was also measured and varied from 36% for toluene-induced P. putida F1 to 102% for M. trichosporium OB3b. Since C₂HCl₃ degradation requires new bio-mass, the specific growth rate (μ_max) of each of the C₂HCl₃-degradation microorganisms was determined and varied from 0.080/h (M. trichosporium OB3b) to 0.864/h (P. cepacia G4 PR1).

Degradation of prochloraz and 2,4,6-trichlorophenol by environmental bacterial strains by C. Bock; R. M. Kroppenstedt; U. Schmidt; H. Diekmann (pp. 257-262).

Eight bacterial isolates from enrichment with 2,4,6-trichlorophenol (TCP) as sole carbon source were tested for their potential to degrade prochloraz. None of them could grow on prochloraz. Strain C964, identified as Aureobacterium sp., effectively reduced the fungitoxic activity of prochloraz in a bioassay and degradation was confirmed by HPLC. Two other isolates, strain C611 and C961, using TCP as a carbon source, belong to the β subclass of the proteobacteria and presumely degrade TCP via 2,4-dichlorohydroquinone and hydroxyhydroquinone as indicated by oxygen-consumption tests.

Influence of environmental parameters on pentachlorophenol biotransformation in soil by Lentinula edodes and Phanerochaete chrysosporium by B. C. Okeke; J. E. Smith; A. Paterson; I. A. Watson-Craik (pp. 263-266).

The influences of temperature, soil moisture potential and initial pH on the biotransformation of pentachlorophenol (PCP) by the lignicolous fungi Lentinula edodes and Phanerochaete chrysosporium were examined. At 10°C, L. edodes was more effective in degrading PCP (P < 0.05) than P. chrysosporium. At 15°C similar results were obtained for the two fungi. The highest levels of degradation occurred for both fungi at 25°C. With P. chrysosporium, the extent of PCP elimination was directly related to soil moisture content and optimal at approximately 47%. With L. edodes, in contrast, the process was inversely related to moisture content and maximal at 26%. The initial soil pH also had a marked influence, and pH 4.0 was optimal for both fungi.

Optimizing biodegradation of phenanthrene dissolved in nonaqueous-phase liquids by I. Birman; M. Alexander (pp. 267-272).

A study was conducted to optimize the biodegradation in soil slurries of phenanthrene initially dissolved in nonaqueous-phase liquids (NAPLs). The slow rate of degradation of phenanthrene in dibutyl phthalate was increased by addition of phenanthrene-degrading microorganisms to soil slurries containing the NAPL. The rate was further increased and the acclimation phase was shortened if the inoculum was grown in a medium containing the hydrocarbon and the phthalate before addition to the slurries. Composition of the growth medium only shortened the acclimation but had no effect on the rate. Vigorous agitation increased the rate and extent of mineralization of phenanthrene in dibutyl phthalate. The effect of temperature was affected by the presence and identity of the inoculum. Rapid and extensive mineralization of phenanthrene initially present in hexadecane and diesel oil were attained by use of intense agitation of the NAPL/soil slurry and inoculation with microorganisms grown in the presence of the NAPLs, but the influence of these variables was less with other NAPLs. Vigorous agitation and addition of an inoculum 24 h after introduction of a nonionic surfactant enhanced biodegradation of phenanthrene initially in 150 Bright stock oil and dibutyl phthalate. The results suggest improved means for the bioremediation of sites contaminated with NAPLs.

Cell-free extract(s) of Pseudomonas putida catalyzes the conversion of cyanides, cyanates, thiocyanates, formamide, and cyanide-containing mine waters into ammonia by G. R. V. Babu; O. K. Vijaya; V. L. Ross; J. H. Wolfram; K. D. Chapatwala (pp. 273-277).

Our isolate, Pseudomonas putida, is known to be capable of utilizing cyanides as the sole source of carbon (C) and nitrogen (N) both in the form of free cells and cells immobilized in calcium alginate. In the present study, the cell-free extract(s) were prepared from the cells of P. putida grown in the presence of sodium cyanide. The ability of enzyme(s) to convert cyanides, cyanates, thiocyanates, formamide and cyanide-containing mine waters into ammonia (NH₃) was studied at pH 7.5 and pH 9.5. The kinetic analysis of cyanide and formamide conversion into NH₃ at pH 7.5 and pH 9.5 by the cell-free extract(s) of P. putida was also studied. The K_m and V_max values for cyanide/formamide were found to be 4.3/8 mM and 142/227 μmol NH₃ released mg protein⁻¹ min⁻¹ respectively at pH 7.5 and 5/16.67 mM and 181/434 μmol NH₃ released mg protein⁻¹ h⁻¹ respectively at pH 9.5. The study thus concludes that the cell-free extract(s) of P. putida is able to metabolize not only cyanides, cyanates, thiocyanates, and formamide but also cyanide-containing mine waters to NH₃.

A method to increase silver biosorption by an industrial strain of Saccharomyces cerevisiae by P. Simmons; I. Singleton (pp. 278-285).

Ag⁺ biosorption by an industrial strain of Saccharomyces cerevisiae was investigated. Older (96 h old) biomass had half the biosorption capacity of younger (24 h old) biomass (0.187 and 0.387 mmol Ag⁺/g dry mass respectively). Comparisons of cell walls isolated from biomass of either age indicated that chemical composition and Ag⁺ biosorption capacity varied little over the time span examined and that cell walls from either age of culture had small Ag⁺ biosorption capacities compared to whole cells of a similar age. Silver-containing precipitates were observed both on the cell wall and within the cell, indicating that intracellular components sorbed Ag⁺. The concentration of these precipitates within the cell appeared visually to decrease with age in Ag⁺-exposed cells. Incorporation of l-cysteine into the growth medium resulted in biomass with increased silver biosorption capacities, protein and sulphydryl group content. Increasing the concentration of l-cysteine in the growth medium from 0 to 5.0 mM increased silver biosorption from 0.389 to 0.556 mmol Ag⁺/g dry mass Isolated cell walls of biomass grown in supplemented media also showed a possible link between silver biosorption capacities, protein and sulphydryl group content. No precipitates were observed in silver-exposed biomass that had been grown in the presence of 5.0 mM l-cysteine.

Influence of physiologically relevant parameters on biomass formation in a trickle-bed bioreactor used for waste gas cleaning by P. Schönduve; M. Sára; A. Friedl (pp. 286-292).

Limitation of biomass formation in a mixed culture immobilised in a trickle-bed bioreactor without substantially affecting the biological degradation of organic compounds in waste gas streams was investigated. As carbon source, the industrially relevant volatile organic compounds ethyl acetate and toluene were used. The temporal biofilm composition was investigated by means of transmission electron microscopy of ultrathin sections cut along the film height. Physiologically relevant parameters were varied. In this context the effect of (a) the type of nitrogen source, (b) the concentration of inert salt and (c) limiting the availability of essential nutrients by intermittent trickling was studied. The effect of these parameters on both biomass formation and degradation was expressed in terms of the ratio R which was defined as the fractional inhibition of biomass formation related to the fractional decrease of degradation. Using nitrate as nitrogen source instead of ammonium, R was 0.71, which means that the fractional inhibition of biomass formation was less than the fractional inhibition of degradation. When the concentration of NaCl as inert salt was adjusted to 0.4 M, the R became 1.32, showing that the fractional inhibition of biomass formation was stronger than the fractional inhibition of degradation. Limiting the availability of nutrients by intermittent trickling, the pressure drop fell by 50% whereas the degradation efficiency decreased by 30%. In summary, intermittent trickling and addition of an inert salt were observed to be advantageous unlike the impact of the type of nitrogen source.

The effect of inoculation and the type of carrier material used on the biofiltration of methyl sulphides by E. Smet; G. Chasaya; H. Van Langenhove; W. Verstraete (pp. 293-298).

Low elimination capacities (less than 10 gm⁻³day⁻¹) were observed for the odorant dimethyl sulphide (Me₂S) when either wood bark or compost was used as the carrier material in a laboratory-scale biofilter. Enrichment experiments were set up by incubation of garden soil samples during 4 weeks with 100 ppm (v/v) headspace concentrations of both Me₂S and dimethyl disulphide (Me₂S₂). After transfer to a mineral medium, Me₂S- and Me₂S₂-degrading enrichment cultures were obtained for all five soil samples tested, both compounds being converted stoichiometrically to sulphuric acid. Upon inoculation of the laboratory-scale biofilter with one of these enrichment cultures (± 120 g cell dry weight m⁻³ reactor), the elimination capacity for Me₂S increased in a 3-week period to 35 gm⁻³ day⁻¹ and 680 gm⁻³ day⁻¹ when wood bark and compost were used as the respective carrier materials. Both inoculated biofilters were able to degrade Me₂S₂, however the elimination capacities obtained for Me₂S₂ were lower (e.g. 24 g m⁻³ day⁻¹ for the wood bark filter) compared to those for Me₂S. For both inoculated biofilters, a gradual decrease of the elimination capacity for the methyl sulphides was observed as a result of acidification of the carrier material, suggesting that pH regulation is necessary if long-term biofiltration experiments are to be performed.

Sections

Personal tools

Applied Microbiology and Biotechnology (v.45, #1-2)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.45, #1-2)