Pyrazine production by Bacillus subtilis in solid-state fermentation on soybeans

2,5-Dimethylpyrazine (2,5-DMP) and tetramethylpyrazine (TTMP) were produced using Bacillus subtilis IFO 3013 grown on soybeans. Solid-state cultivations were carried out either in 100-ml bottles or in a fixed-bed column reactor, both systems being at 27 °C. Optimization studies showed that the best way to produce the two above aroma compounds involved two separate processes. 2,5-DMP was obtained using soybeans enriched with 75 g threonine/kg initial dry weight (i.d.w.), giving 0.85 g metabolite/kg i.d.w. after 6 days. TTMP production involved addition of 90 g/kg i.d.w. acetoin to soybeans, and 2.5 g/kg i.d.w. was recovered after 14 days. These results demonstrated the suitability of solid-state cultivation for production of high-added-value compounds. Received: 30 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996     

Purification of α-amylase by specific elution from anti-peptide antibodies

Chimeric α-amylase, produced by recombinant yeast cells, was purified by immunoaffinity chromatography by use of an anti-peptide antibody and an eluent containing an antigen peptide. Chimeric α-amylase was adsorbed by the antibody against the peptide corresponding to the C-terminal region of target α-amylase, and specifically eluted by the eluent containing the antigen peptide used for immunization. A low concentration of the peptide could competitively elute adsorbed α-amylase, and the rate-limiting step of the elution was mass transfer of desorbed α-amylase. With this specific method, target proteins can be effectively eluted, and highly purified under mild conditions, from the antibody ligand showing a high-affinity for the adsorption step. Received: 14 November 1996 / Received revising: 16 December 1996 / Accepted: 17 January 1997     

Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [¹⁴C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [¹⁴C]TNT (100 μM corresponding to 4.75 μCi/l) to ¹⁴CO₂ within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [¹⁴C]TNT significantly. Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Effect of a biofilm on the adsorption of 4-chlorophenol on activated carbon

  The adsorption of 4-chlorophenol (4-CP) on activated carbon was studied experimentally both in the presence and in the absence of an inactivated anaerobic biofilm on the surface of carbon pellets. The presence of the biofilm markedly decreased the rate of 4-CP adsorption. However, the final near-equilibrium state (at 27 h) was not affected, and the incremental amount of material adsorbed on the pellets was similar both in the presence and in the absence of the biofilm. The biosorption of 4-CP by a biofilm coating non-adsorbing pellets was also determined. It appears that the biofilm also has some adsorption capability. Freundlich-type equations were used to correlate all data, and transient and near-equilibrium isotherms were obtained for 4-CP adsorption on different adsorbing materials at different times. Received: 30 April 1996 / Received revision: 17 July 1996 / Accepted: 4 August 1996     

Axenic aerobic biofilms inhibit corrosion of SAE 1018 steel through oxygen depletion

Corrosion inhibition of SAE 1018 steel by pure-culture biofilms of Pseudomonas fragi and Escheri-chia coli DH5α has been evaluated in complex Luria-Bertani medium, seawater-mimicking medium, and modified Baar's medium at 30 °C. In batch cultures, both bacteria inhibited corrosion three to six fold compared to sterile controls, and the corrosion was comparable to that observed in anaerobic sterile media. To corroborate this result, a continuous reactor and electrochemical impedance spectroscopy were used to show that both P. fragi K and E. coli DH5α decreased the corrosion rate by 4- to 40-fold as compared to sterile controls; this matched the decrease in corrosion found with sterile medium in the absence of oxygen and with E. coli DH5α grown anaerobically. In addition, the requirement for live respiring cells was demonstrated by the increase in the corrosion rate that was observed upon killing the P. fragi K biofilm in continuous cultures, and it was shown that fermentation products do not cause an increase in corrosion. Hence, pure-culture biofilms inhibit corrosion of SAE 1018 steel by depleting oxygen at the metal surface. Received: 16 December 1996 / Received revision: 18 March 1997 / Accepted: 27 March 1997     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. III.In vitro depolymerization of coal humic acids by a crude preparation of manganese peroxidase from the white-rot fungus Nematoloma frowardii b19

The in vitro depolymerization of humic acids derived from German lignite (low-rank coal, brown coal) was studied using a manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii b19. The H₂O₂ required was continuously generated by glucose oxidase. Mn peroxidase depolymerized high-molecular-mass humic acids by forming fulvic-acid-like compounds. The depolymerization process was accompanied by the decolorization of the dark-brown humic acid fraction soluble in alkaline solutions (decrease in absorbance at 450 nm) and by the yellowish coloring of the fraction of acid-soluble fulvic-acid-like compounds (increase in absorbance at 360 nm). The Mn peroxidase of N. frowardii b19 has been proved to be highly stable; even after an in vitro reaction time of 7 days in the presence of humic acids, less than 10% loss in total oxidizing activity was detectable. Received: 16 September 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

The lamellar settler – a low-cost alternative for separating the micro-alga Chlorella vulgaris from a cultivation broth?

Micro-algae, especially Chlorella vulgaris, produce a range of high-value substances and the biomass itself is used for purposes such as feeding in aquaculture. A lamellar settler was designed and built. Its suitability as a low-cost alternative to separate C. vulgaris was investigated. The settler operated semicontinuously in a laboratory photoreactor plant (total volume 9 l). A clearing of 30%–35% and a 50% increase in harvest outflow concentration were observed. The scaled up data for design and construction of a settler for a 200-l production plant were elaborated. Received: 25 September 1996 / Received revision: 10 December 1996 / Accepted: 15 December 1996     

Production, purification, and characterization of a highly enantioselective (S )-N-acetyl-1-phenylethylamine amidohydrolase from Rhodococcus equi Ac6

Rhodococcus equi Ac6 was found to express an inducible (S )-specific N-acetyl-1-phenylethylamine amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed around pH 6.5. Purification of the enzyme to a single band in a Coomassie-blue-stained sodium dodecyl sulfate/polyacrylamide gel (SDS-PAGE) was achieved by ammonium sulfate precipitation of R. equi Ac6 crude extract and column chromatographies on Fractogel TSK Butyl-650(S) and Superose 12HR. At pH 7.0 and 30 °C the amidohydrolase had a half-life of around 350 days; at 44 °C it was only 10 min. Except for Ni²⁺ and, to some extent, Zn²⁺ and Co²⁺, the enzyme was neither strongly influenced by metal cations nor by chelating agents, but was inhibited by 95% at 0.1 mM phenylmethylsulfonyl fluoride. The molecular mass of the native enzyme was estimated to be 94 kDa by gel filtration and 50 kDa by SDS-PAGE, suggesting a dimeric structure. Specificity experiments revealed a spectrum of related N-acetylated compounds being hydrolyzed with variable enantiomeric selectivities. Received: 20 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996     

Isolation of isoflavones from soy-based fermentations of the erythromycin-producing bacterium Saccharopolyspora erythraea

A search for an abundant and economical source of isoflavones, particularly genistein, led to the discovery that the erythromycin-producing organism Saccharopolyspora erythraea also produces this promising new cancer-prevention agent. Erythromycin fermentation is a large-scale, soybean-based process used world-wide for the commercial production of this medically important antibiotic. Results from this study indicate that genistin (the glucoside form of genistein), which is added to the fermentation in the soybean media, was converted to genistein through the action of a β-glucosidase produced by the organism. Genistein was co-extracted with erythromycin from the fermentation broth, then separated from erythromycin during the second step of the purification process for the production of erythromycin. Received 10 September 1996 / Received revision: 22 November 1996 / Accepted: 7 December 1996     

Enhanced oily sludge biodegradation by a tensio-active agent isolated from Pseudomonas aeruginosa USB-CS1

The biodegradation of an oily sludge is facilitated by a microbial tensio-active agent isolated from Pseudomonas aeruginosa USB-CS1. The optimal oil-in-water dispersion conditions are as follows: pH 6.5, temperature 30 °C, agitation 150 rev/min. The total hydrocarbon content shows that the biodegradation of the oily substrate mediated by the biosurfactant or by the biosurfactant–P. aeruginosa USB-CS1 complex is significantly higher after 30 days of incubation than that in other experimental conditions, by a mean of 70%. Substrate fractionation by column chromatography reveals that, if biosurfactant is present, saturated and aromatic compounds are more susceptible to microbial degradation than they are in other biodegradation systems by an average of 55% and 40% respectively. These results suggest that the stimulatory effects of the biosurfactant on the biodegradation of the oily substrate are limited over time by the loss of surface activity of the biosurfactant after 30 days of incubation. Received : 7 August 1996 / Received revision : 6 December 1996 / Accepted : 4 January 1997     

Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b

A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8 and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity was inhibited by Cu²⁺, Co²⁺, Ni²⁺, and Zn²⁺, and this inhibition was reversed by EDTA.M Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996     

A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan

An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides. Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996     

Phycocyanin,a new elicitor for capsaicin and anthocyanin accumulation in plant cell cultures

  Elicitors of both fungal and bacterial origin that is, polysaccharides, proteins and fatty acids, are widely used for enhancement of secondary metabolites in plant cell cultures. In the present study, phycocyanin – a natural blue pigment that is the major light-harvesting biliprotein in the blue-green alga Spirulina platensis– was used as an elicitor to enhance the accumulation of capsaicin and anthocyanin in Capsicum frutescens and Daucus carota cell cultures respectively. Phycocyanin at 0.3, 0.6 and 1.2 mg% in capsicum cell cultures elicited a more than two-fold increase in capsaicin content with maximum productivity of 192 μg/g fresh weight. Similarly in Daucus carota cell cultures a two-fold increase in anthocyanin content was obtained at 0.3 mg% with a maximum productivity of 24.8 mg% on a dry-weight basis. In both the systems, phycocyanin showed an early elicitation of secondary metabolites. Received: 15 December 1995 / Received last revision: 15 July 1996 / Accepted: 18 July 1996     

Cell-dislodging methods under serum-free conditions

In this work, a BHK21 clone producing a fusion protein consisting of a recombinant human IgG molecule with a cytokine tail, growing in a protein-free medium, was used to test several alternatives to avoid the use of serum for trypsin inactivation, currently used in cell dislodging. These included (1) trypsin inactivated with soybean trypsin inhibitor (STI); (2) cell dissociation solution instead of trypsin; (3) dispase instead of trypsin; (4) trypsin inactivated with fetal calf serum (positive control); (5) non-inactivated trypsin (negative control). Use of a centrifugation step was also tested for each alternative. Results indicate that the best method regarding cell growth, viability and adherent fraction is to use trypsin inactivated with STI followed by a centrifugation step. For all methods tested, the utilization of a centrifugation step always led to improved results. The optimal proportion for total trypsin inactivation is 1:1 trypsin (0.2% w/v) to STI (1 mg ml⁻¹), equivalent to 2 mg trypsin to 1 mg STI. No toxic effect was observed for STI at the concentrations used. Long-term subculturing with this new, alternative dislodging method did not affect cell growth, viability and productivity. Received: 23 September 1996 / Received revision: 27 December 1996 / Accepted: 30 December 1996     

Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol

An electrochemical reactor employing activated carbon fibers (ACF) was constructed for the disinfection of bacteria in drinking water. The application of an alternating potential of 1.0 V and −0.8 V versus a saturated calomel electrode, for disinfecting and desorbing bacteria, enabled reactor operation for 840 h. Drinking water was passed through the reactor in stop/flow mode: 300 ml/min flow for 12 h and no flow for 12 h, alternately. The bacterial cell density in treated water was always been less than 20 cells/ml. It was also found that the formation of biofilm on the ACF reactor caused an increase in current, enabling the self-detection of microbial fouling. Received: 19 February 1996 / Received last revision: 23 July 1996 / Accepted: 2 September 1996     

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997     

Corrosion inhibition by aerobic biofilms on SAE 1018 steel

Carbon steel (SAE 1018) samples were exposed to complex liquid media containing either the aerobic bacterium Pseudomonas fragi or the facultative anaerobe Escherichia coli DH5α. Compared to sterile controls, mass loss was consistently 2- to 10-fold lower in the presence of these bacteria which produce a protective biofilm. Increasing the temperature from 23 °C to 30 °C resulted in a 2- to 5-fold decrease in corrosion inhibition with P. fragi whereas the same shift in temperature resulted in a 2-fold increase in corrosion inhibition with E. coli DH5α. Corrosion observed with non-biofilm-forming Streptomyces lividans TK24 was similar to that observed in sterile media. A dead biofilm, generated in situ by adding kanamycin to an established biofilm, did not protect the metal (corrosion rates were comparable to those in the sterile control), and mass loss in cell-free, spent Luria-Bertani (LB) medium was similar to that in sterile medium. Confocal laser scanning microscopy analysis confirmed the presence of a biofilm consisting of live and dead cells embedded in a sparse glycocalyx matrix. Mass-loss measurements were consistent with microscopic observations of the metal surface after 2 weeks of exposure, indicating that uniform corrosion occurred. The biofilm was also able to withstand mild agitation (60 rpm), provided that sufficient time was given for its development. Received: 3 May 1996 / Received revision: 8 August 1996 / Accepted: 24 August 1996     

Transformation of tetrachloroethene in an upflow anaerobic sludgeblanket reactor

  Reductive dechlorination of tetrachloroethene was studied in a mesophilic upflow anaerobic sludge blanket reactor. Operating the reactor in batch mode the dynamic transformation of tetrachloroethene, trichloroethene and dichloroethene (DCE) was monitored. Tetrachloroethene was reductively dechlorinated to trichloroethene, which again was dechlorinated at the same rate as DCE was produced. DCE showed a lag period of 40 h before transformation was observed. During normal reactor operation trans-1,2-DCE was the major DCE isomer, followed by cis-1,2-DCE. Small amounts of 1,1-DCE but no vinyl chloride were detected. When the influent tetrachloroethene concentration was increased from 4.6 μM to 27 μM, the transformation rate increased, indicating that the system was not saturated with tetrachloroethene. The main organic component in the effluent was acetate, indicating that the aceticlastic methane-producing bacteria were inhibited by the chlorinated ethenes. Received: 29 July 1996 / Received revision: 13 September 1996 / Accepted: 13 September 1996     

Influence of aeration and carbon source on production of microcin B17 by Escherichia coli ZK650

Previous studies [Connell et al. (1987) Mol Microbiol 1: 195–201] have shown that expression of the microcin B17 (MccB17) promoter is inversely related to the growth rate of the culture, when slower growth was brought about by limitation of sources of carbon, nitrogen or phosphorus. When we used oxygen limitation to decrease growth in a glucose-based chemically defined medium, we found specific MccB17 production to be positively related to growth rate and extent. On the other hand, when we examined various nutritional variations of media, specific production of MccB17 showed a negative relationship to growth rate and extent, as would be predicted by the findings of Connell et al. (1987). Glucose, glycerol and acetate were found to repress MccB17 production; succinate was not repressive. Succinate is an excellent carbon source for production of MccB17 since high levels can be used with no or little interference in product synthesis. Received: 26 November 1996 / Accepted: 7 December 1996     

Heterologous gene expression of bovine plasmin in Lactococcus lactis

Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin produced was shown to be biologically active. Received: 2 December 1996 / Received revision: 17 March 1997 / Accepted: 27 April 1997